Methods of treating pediatric cancers

ABSTRACT

A method of treating a pediatric cancer in a subject in need thereof. The method includes administering to the subject a therapeutically effective amount of (S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide, or a pharmaceutically acceptable salt thereof, or a combination thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to PCT/US2017/025932, filed Apr. 4,2017; which claims priority to U.S. Provisional Application Ser. No.62/318,041, filed Apr. 4, 2016; 62/323,437, filed Apr. 15, 2016;62/329,653, filed Apr. 29, 2016; 62/380,773, filed Aug. 29, 2016; and62/449,366, filed Jan. 23, 2017, each of which is incorporated byreference in its entirety herein.

BACKGROUND 1. Field of the Invention

The present disclosure relates to(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide(Formula (I)) and to pharmaceutically acceptable salts thereof, forexample the hydrogen sulfate salt, a crystalline form of the hydrogensulfate salt, and further to liquid formulations thereof, which exhibitTrk family protein tyrosine kinase inhibition, and the use of thecompound, salts, crystalline forms, and liquid formulations in thetreatment of pediatric cancers.

2. Description of the Related Art

Infantile fibrosarcoma (IFS) is a rare pediatric cancer typicallypresenting in the first two years of life. Surgical resection can becurative and chemotherapy is active against gross residual disease.However, when recurrences occur, therapeutic options are often limited.

Trk's are the high affinity receptor tyrosine kinases activated by agroup of soluble growth factors called neurotrophins (NT). The Trkreceptor family has three members—TrkA, TrkB and TrkC. Among theneurotrophins are (i) nerve growth factor (NGF) which activates TrkA,(ii) brain-derived neurotrophic factor (BDNF) and NT-4/5 which activateTrkB and (iii) NT3 which activates TrkC. Trk's are widely expressed inneuronal tissue and are implicated in the maintenance, signaling andsurvival of neuronal cells (Patapoutian, A. et al., Current Opinion inNeurobiology, 2001, 11, 272-280).

Recent literature has shown that overexpression, activation,amplification and/or mutation of Trk's are associated with many cancersincluding neuroblastoma (Brodeur, G. M., Nat. Rev. Cancer 2003, 3,203-216), ovarian cancer (Davidson., B. et al., Clin. Cancer Res. 2003,9, 2248-2259), breast cancer (Kruettgen et al., Brain Pathology 2006,16: 304-310), prostate cancer (Dionne et al., Clin. Cancer Res. 1998,4(8): 1887-1898), pancreatic cancer (Dang et al., Journal ofGastroenterology and Hepatology 2006, 21(5): 850-858), multiple myeloma(Hu et al., Cancer Genetics and Cytogenetics 2007, 178: 1-10),astrocytoma amd medulloblastoma (Kruettgen et al., Brain Pathology 2006,16: 304-310), glioma (Hansen et al., Journal of Neurochemistry 2007,103: 259-275), melanoma (Nakagawara, A. (2001) Cancer Letters169:107-114; Meyer, J. et al. (2007) Leukemia, 1-10; Pierottia, M. A.and Greco A., (2006) Cancer Letters 232:90-98; Eric Adriaenssens, E. etal. Cancer Res (2008) 68:(2) 346-351), thyroid carcinoma (Brzezianska etal., Neuroendocrinology Letters 2007, 28(3), 221-229), lungadenocarcinoma (Perez-Pinera et al., Molecular and Cellular Biochemistry2007, 295(1&2), 19-26), large cell neuroendocrine tumors (Marchetti etal., Human Mutation 2008, 29(5), 609-616), and colorectal cancer(Bardelli, A., Science 2003, 300, 949). In preclinical models of cancer,Trk inhibitors are efficacious in both inhibiting tumor growth andstopping tumor metastasis. In particular, non-selective small moleculeinhibitors of TrkA, TrkB, TrkC and Trk/Fc chimeras were efficacious inboth inhibiting tumor growth and stopping tumor metastasis (Nakagawara,A. (2001) Cancer Letters 169:107-114; Meyer, J. et al. (2007) Leukemia,1-10; Pierottia, M. A. and Greco A., (2006) Cancer Letters 232:90-98;Eric Adriaenssens, E. et al. Cancer Res (2008) 68:(2) 346-351).Therefore, an inhibitor of the Trk family of kinases is expected to haveutility in the treatment of cancer.

In addition, inhibitors of the Trk/neurotrophin pathway have beendemonstrated to be effective in numerous pre-clinical animal models ofpain. For example, antagonistic NGF and TrkA antibodies (for example,RN-624) have been shown to be efficacious in inflammatory andneuropathic pain animal models and in human clinical trials (Woolf, C.J. et al. (1994) Neuroscience 62, 327-331; Zahn, P. K. et al. (2004),Pain 5, 157-163; McMahon, S. B. et al., (1995) Nat. Med. 1, 774-780; Ma,Q. P. and Woolf, C. J. (1997) Neuroreport 8, 807-810; Shelton, D. L. etal. (2005) Pain 116, 8-16; Delafoy, L. et al. (2003) Pain 105, 489-497;Lamb, K. et al. (2003) Neurogastroenterol. Motil. 15, 355-361; Jaggar,S. I. et al. (1999) Br. J. Anaesth. 83, 442-448). Additionally, recentliterature indicates after inflammation. BDNF levels and TrkB signalingis increased in the dorsal root ganglion (Cho, L. et al. Brain Research1997, 749, 358) and several studies have shown antibodies that decreasesignaling through the BDNF/TrkB pathway inhibit neuronalhypersensitization and the associated pain (Chang-Qi, L et al. MolecularPain 2008, 4:27).

It has been shown that NGF secreted by tumor cells and tumor invadingmacrophages directly stimulates TrkA located on peripheral pain fibers.Using various tumor models in both mice and rats it was demonstratedthat neutralizing NGF with a monoclonal antibody inhibits cancer relatedpain to a degree similar or superior to the highest tolerated dose ofmorphine. In addition, activation of the BDNF/TrkB pathway has beenimplicated in numerous studies as a modulator of various types of painincluding inflammatory pain (Matayoshi, S., J. Physiol. 2005,569:685-95), neuropathic pain (Thompson, S. W., Proc. Natl. Acad. Sci.USA 1999, 96:7714-18) and surgical pain (Li, C.-Q. et al., MolecularPain, 2008, 4(28), 1-11). Because TrkA and TrkB kinases may serve as amediator of NGF driven biological responses, inhibitors of TrkA and/orother Trk kinases may provide an effective treatment for chronic painstates.

The current treatment regimens for pain conditions utilize severalclasses of compounds. The opioids (such as morphine) have severaldrawbacks including emetic, constipatory and negative respiratoryeffects, as well as the potential for addictions. Non-steroidalanti-inflammatory analgesics (NSAIDs, such as COX-1 or COX-2 types) alsohave drawbacks including insufficient efficacy in treating severe pain.In addition, COX-1 inhibitors can cause ulcers of the mucosa.Accordingly, there is a continuing need for new and more effectivetreatments for the relief of pain, especially chronic pain.

In addition, inhibition of the neurotrophin/Trk pathway has been shownto be effective in treatment of pre-clinical models of inflammatorydiseases. For example, inhibition of the neurotrophin/Trk pathway hasbeen implicated in preclinical models of inflammatory lung diseasesincluding asthma (Freund-Michel, V; Frossard, N.; Pharmacology &Therapeutics (2008), 117(1), 52-76), interstitial cystitis (Hu Vivian Y;et. al. The Journal of Urology (2005), 173(3), 1016-21), inflammatorybowel diseases including ulcerative colitis and Crohn's disease (DiMola, F. F, et. al., Gut (2000), 46(5), 670-678) and inflammatory skindiseases such as atopic dermatitis (Dou, Y.-C.; et. al. Archives ofDermatological Research (2006), 298(1), 31-37), eczema and psoriasis(Raychaudhuri, S. P.; et. al. Journal of Investigative Dermatology(2004), 122(3), 812-819).

The neurotrophin/Trk pathway, particularly BDNF/TrkB, has also beenimplicated in the etiology of neurodegenerative diseases includingmultiple sclerosis, Parkinson's disease and Alzheimer's disease(Sohrabji, Farida; Lewis, Danielle K. Frontiers in Neuroendocrinology(2006), 27(4), 404-414). Modulation of the neutrophin/Trk pathway mayhave utility in treatment of these and related diseases.

The TrkA receptor is also thought to be critical to the disease processin the infection of the parasitic infection of Trypanosoma cruzi (Chagasdisease) in human hosts (de Melo-Jorge, M. et al. Cell Host & Microbe(2007), 1(4), 251-261). Thus, TrkA inhibition may have utility intreating Chagas disease and related protozoan infections.

Trk inhibitors may also find use in treating disease related to animbalance of the regulation of bone remodeling, such as osteoporosis,rheumatoid arthritis, and bone metastases. Bone metastases are afrequent complication of cancer, occurring in up to 70 percent ofpatients with advanced breast or prostate cancer and in approximately 15to 30 percent of patients with carcinoma of the lung, colon, stomach,bladder, uterus, rectum, thyroid, or kidney. Osteolytic metastases cancause severe pain, pathologic fractures, life threatening hypercalcemia,spinal cord compression, and other nerve-compression syndromes. Forthese reasons, bone metastasis is a serious and costly complication ofcancer. Therefore, agents that can induce apoptosis of proliferatingosteoblasts would be highly advantageous. Expression of TrkA and TrkCreceptors has been observed in the bone forming area in mouse models ofbone fracture (K. Asaumi, et al., Bone (2000) 26(6) 625-633). Inaddition, localization of NGF was observed in almost all bone formingcells (K. Asaumi, et al.). Recently, it was demonstrated that a pan-Trkinhibitor inhibits the tyrosine signaling activated by neurotrophinsbinding to all three of the Trk receptors in human hFOB osteoblasts (J.Pinski, et al., (2002) 62, 986-989). These data support the rationalefor the use of Trk inhibitors for the treatment of bone remodelingdiseases, such as bone metastases in cancer patients.

Several classes of small molecule inhibitors of Trk kinases said to beuseful for treating pain or cancer are known (Expert Opin. Ther. Patents(2009) 19(3)).

International Patent Application Publications WO 2006/115452 and WO2006/087538 describe several classes of small molecules said to beinhibitors of Trk kinases which could be useful for treating pain orcancer.

Pyrazolo[1,5-a]pyrimidine compounds are known. For example,International Patent Application Publication WO 2008/037477 disclosespyrazolo[1,5-a]pyrimidine compounds bearing an alkyl, aryl orheterocyclic group at the 3-position. These compounds are asserted to bePI3K and/or mTOR Lipid Kinase inhibitors.

PCT Patent Publication No. WO 2008/058126 disclosespyrazolo[1,5-a]pyrimidine compounds bearing a phenyl group at the3-position. These compounds are asserted to be Pim-kinase inhibitors.

U.S. Patent Publication No. 2006/0094699 disclosespyrazolo[1,5-a]pyrimidine compounds bearing a —C(═O)NH-phenyl,—C(═O)(4-methylpiperidinyl) or —C(═O)NMe(CH₂-trimethylpyrazolyl) groupat the 3-position for use in combination therapy with a glucocorticoidreceptor agonist.

PCT Patent Publication Nos. WO 2010/033941, WO 2010/048314, WO2011/006074, and WO 2011/146336 disclose compounds which exhibit Trkfamily protein tyrosine kinase inhibition, and which are useful in thetreatment of pain, cancer, inflammation, neurodegenerative diseases andcertain infectious diseases.

WO 2010/048314 discloses in Example 14A a hydrogen sulfate salt of(S)—N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide.WO 2010/048314 does not disclose the particular form of the hydrogensulfate salt described herein when prepared according to the method ofExample 14A in that document. In particular, WO 2010/048314 does notdisclose crystalline form (l-HS) as described below.

All documents, including scientific articles, patent publications andapplications, and the like, referenced in the present disclosure arehereby incorporated by reference in their entirety.

SUMMARY

Provided herein are methods of treating a pediatric cancer in a subjectin need thereof comprising administering to the subject atherapeutically effective amount of(S)—N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehaving the formula (I):

a pharmaceutically acceptable salt thereof, or combinations thereof.

In some embodiments, the subject is an infant, child, or adolescent. Forexample, the subject is an infant.

In some embodiments, the pediatric cancer is a mesenchymal cancer. Forexample, the mesenchymal cancer can be selected from the groupconsisting of: pediatric nephroma, congenital fibrosarcoma (CFS),pediatric high-grade glioma (HGG), mesenchymal cancers (infantfibrosarcoma (IF), congenital mesoblastic nephroma, congenital infantilefibrosarcoma (CIFS); pilocytic astrocytoma, brain tumors, pediatic acuteleukemia, Ph-like acute lymphoblastic leukemia, cellular congenitalmesoblastic nephroma (CMN); infantile fibrosarcoma, pediatric high-gradeglioma (HGG), diffuse intrinsic pontine gliomas (DIPGs), non-brainstemHGGs (NBS-HGGs), anaplastic large cell lymphoma (ALCL), non-Hodgkin'slymphoma (NHL), pediatric papillary thyroid carcinoma, soft tissuesarcoma, spitzoid melanoma, pediatric hemangiopericytoma-like sarcoma,spindle cell sarcoma, NOS with myo/haemangiopericytic growth pattern,lung cancer, advanced pediatric solid tumors, neuroectodermal-derivedtumors, pediatric colorectal cancer, adrenal neuroblastoma, and centralnervous system tumors.

In some embodiments, the pediatric cancer is a fibrosarcoma such asinfantile fibrosarcoma.

In some embodiments, the pediatric cancer is a glioma. For example, thepediatric cancer is selected from the group consisting of: pediatrichigh-grade glioma (HGG), diffuse intrinsic pontine gliomas (DIPGs), andon-brainstem HGGs (NBS-HGGs).

In some embodiments, the pediatric cancer is an extracranial solidtumor. For example, the pediatric cancer is selected from the groupconsisting of: neuroblastoma, nephroblastoma (e.g., Wilm's tumor),rhabdomyosarcoma and hepatoblastoma

In some embodiments, the cancer is mediated by TrkA. In someembodiments, the cancer is mediated by TrkB. In some embodiments, thecancer is mediated by TrkC. In some embodiments, the cancer is mediatedby TrkA, TrkB, TrkC, or combinations thereof.

In some embodiments, surgical resection has failed to inhibitprogression of the fibrosarcoma in the subject. In some embodiments,chemotherapy has failed to inhibit tumor progression in the subject. Insome such embodiments, the chemotherapy comprises administering at leastone of vincristine, actinomycin-D, cyclophosphamide, ifosfamide,etoposide, or doxorubicin. For example, the chemotherapy includingadministering vincristine, actinomycin-D, and cyclophosphamide hasfailed to inhibit tumor progression in the subject. In some embodiments,the chemotherapy including administering ifosfamide and doxorubicin hasfailed to inhibit tumor progression in the subject.

In some embodiments, the subject has a cancer that is ETV6-NTRK3 fusionpositive.

In some embodiments, the methods provided herein further includeperforming a morphological diagnosis, molecular testing, or both priorto administering the compound of formula (I), a pharmaceuticallyacceptable salt thereof, or a combination thereof.

Also provided herein is a method of treating an infantile fibrosarcoma,the method comprising administering to the subject a therapeuticallyeffective amount of(S)—N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehaving the formula (I):

a pharmaceutically acceptable salt thereof, or a combination thereof.

Further provided herein is a method of treating a subject diagnosed oridentified as having a Trk-associated pediatric cancer, the methodcomprising administering to the subject a therapeutically effectiveamount of(S)—N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide having theformula (I):

a pharmaceutically acceptable salt thereof, or a combination thereof.

This disclosure also provides a method for treating a pediatric cancerin a subject in need thereof, the method comprising:

(a) determining if the cancer is associated with one or more ofoverexpression, activation, amplification, and mutation of a Trk kinase;and

(b) if the cancer is determined to be associated with one or more ofoverexpression, activation, amplification, and mutation of a Trk kinase,administering to the subject a therapeutically effective amount of(S)—N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide having theformula (I):

a pharmaceutically acceptable salt thereof, or a combination thereof.

This disclosure also provides a method for treating a pediatric cancerin a subject in need thereof, the method comprising:

(a) identifying the cancer as associated with one or more ofoverexpression, activation, amplification, and mutation of a Trk kinase;and

(b) administering to the subject a therapeutically effective amount of(S)—N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidin-1-yl)-pyrazolo[15-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehaving the formula (I):

a pharmaceutically acceptable salt thereof, or a combination thereof.

Further provided herein are methods for treating a pediatric cancer in asubject in need thereof, the method comprising:

(a) determining if the cancer is mediated by a Trk kinase; and

(b) if the cancer is determined to be mediated by a Trk kinase,administering to the subject a therapeutically effective amount of(S)—N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehaving the formula (I):

a pharmaceutically acceptable salt thereof, or a combination thereof.

Further provided herein are methods for treating a pediatric cancer in asubject in need thereof, the method comprising:

(a) identifying the cancer as mediated by a Trk kinase; and

(b) administering to the subject a therapeutically effective amount of(S)—N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide

having the formula (I):

a pharmaceutically acceptable salt thereof, or a combination thereof.

Also provided herein are methods of treating a pediatric subjectcomprising:

(a) performing an assay on a sample obtained from the subject todetermine whether the subject has dysregulation of a NTRK gene, a Trkprotein, or expression or level of the same; and

(b) administering to a subject determined to have dysregulation of aNTRK gene, a Trk protein, or expression or activity, or level of thesame a therapeutically effective amount of(S)—N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehaving the formula (I):

a pharmaceutically acceptable salt thereof, or a combination thereof. Insome embodiments, the dysregulation of a NTRK gene, a Trk protein, orexpression or level of the same is a chromosome translation that resultsin the translation of a Trk fusion protein. For example, the Trk fusionprotein can be NTRK3-ETV6. In some embodiments, the dyregulation of aNTRK gene, a Trk protein, or expression or activity of the same is oneor more point mutation in the gene.

In some embodiments provided herein, the compound of formula (I) is apharmaceutically acceptable salt. For example, the compound of formula(I) can be a hydrogen sulfate salt. In some embodiments, the compound offormula (I) is provided as a crystalline form. For example, thecrystalline form can have the formula (I-HS):

In some embodiments, the compound of formula (I), a pharmaceuticallyacceptable salt thereof, or a combination thereof, is provided as aliquid formulation. In some embodiments, the compound of formula (I), apharmaceutically acceptable salt thereof, or a combination thereof, ispresent in the liquid formulation an amount from about 0.5 wt. % toabout 7 wt. %. For example, the compound of formula (I), apharmaceutically acceptable salt thereof, or a combination thereof canbe present in the liquid formulation in an amount of about 1.5 wt. % toabout 2.5 wt. %.

In some embodiments, the compound of formula (I), the pharmaceuticallyacceptable salt thereof, or the combination thereof, has a concentrationof about 5 mg/mL to about 50 mg/mL in the liquid formulation. Forexample, the compound of formula (I), the pharmaceutically acceptablesalt thereof, or the combination thereof, can have a concentration ofabout 15 mg/mL to about 35 mg/mL in the liquid formulation. In someembodiments, the compound of formula (I), the pharmaceuticallyacceptable salt thereof, or the combination thereof, has a concentrationof about 20 mg/mL in the liquid formulation.

The solubilizing agent can be selected from the group consisting of acyclodextrin, a glycol, a glycerol, and combinations thereof. In someembodiments, the solubilizing agent includes a cyclodextrin. Forexample, the solubilizing agent can be selected from the groupconsisting of a β-cyclodextrin derivative, a γ-cyclodextrin, andcombinations thereof. In some embodiments, the solubilizing agentincludes a hydroxy alkyl-γ-cyclodextrin. The solubilizing agent caninclude a β-cyclodextrin selected from the group consisting of a hydroxyalkyl-β-cyclodextrin, a sulfoalkyl ether-β-cyclodextrin, andcombinations thereof. In some embodiments, the solubilizing agentincludes hydroxypropyl-β-cyclodextrin.

In some embodiments, the solubilizing agent is present in the liquidformulation in an amount of about 5 wt. % to about 35 wt. %. Forexample, the solubilizing agent can be present in the liquid formulationin an amount of about 13 wt. % to about 17 wt. %.

The liquid formulation can further include a buffer. In someembodiments, the buffer includes at least one of a citrate buffer, alactate buffer, a phosphate buffer, a maleate buffer, a tartaratebuffer, a succinate buffer, or an acetate buffer. In some embodiments,the buffer includes at least one of lithium lactate, sodium lactate,potassium lactate, calcium lactate, lithium phosphate, sodium phosphate,potassium phosphate, calcium phosphate, lithium maleate, sodium maleate,potassium maleate, calcium maleate, lithium tartarate, sodium tartarate,potassium tartarate, calcium tartarate, lithium succinate, sodiumsuccinate, potassium succinate, calcium succinate, lithium acetate,sodium acetate, potassium acetate, or calcium acetate. The buffer can bea citrate buffer. The citrate buffer can include at least one of lithiumcitrate monohydrate, sodium citrate monohydrate, potassium citratemonohydrate, calcium citrate monohydrate, lithium citrate dihydrate,sodium citrate dihydrate, potassium citrate dihydrate, calcium citratedihydrate, lithium citrate trihydrate, sodium citrate trihydrate,potassium citrate trihydrate, calcium citrate trihydrate, lithiumcitrate tetrahydrate, sodium citrate tetrahydrate, potassium citratetetrahydrate, calcium citrate tetrahydrate, lithium citratepentahydrate, sodium citrate pentahydrate, potassium citratepentahydrate, calcium citrate pentahydrate, lithium citrate hexahydrate,sodium citrate hexahydrate, potassium citrate hexahydrate, calciumcitrate hexahydrate, lithium citrate heptahydrate, sodium citrateheptahydrate, potassium citrate heptahydrate, or calcium citrateheptahydrate.

In some embodiments, the buffer includes at least one of sodium citratemonohydrate, potassium citrate monohydrate, calcium citrate monohydrate,sodium citrate dihydrate, potassium citrate dihydrate, calcium citratedihydrate, sodium citrate trihydrate, potassium citrate trihydrate,calcium citrate trihydrate, sodium citrate tetrahydrate, potassiumcitrate tetrahydrate, calcium citrate tetrahydrate, sodium citratepentahydrate, potassium citrate pentahydrate, calcium citratepentahydrate, sodium citrate hexahydrate, potassium citrate hexahydrate,calcium citrate hexahydrate, sodium citrate heptahydrate, potassiumcitrate heptahydrate, or calcium citrate heptahydrate.

In some embodiments, the buffer includes sodium citrate dihydrate.

The buffer can be present in the liquid formulation in an amount ofabout 0.1 wt. % to about 5 wt. %.

In some embodiments, the formulation has a pH of about 2 to about 7. Forexample, the formulation can have a pH of about 3 to about 4. In someembodiments, the formulation has a pH of about 3.5.

In some embodiments, the pH of the liquid formulation is adjusted. Insome such embodiments, the formulation includes a base. For example, thebase can include one or more of a citrate, a lactate, a phosphate, amaleate, a tartarate, a succinate, an acetate, a carbonate, and ahydroxide. In some embodiments, the formulation includes at least one oflithium lactate, sodium lactate, potassium lactate, calcium lactate,lithium phosphate, sodium phosphate, potassium phosphate, calciumphosphate, lithium maleate, sodium maleate, potassium maleate, calciummaleate, lithium tartarate, sodium tartarate, potassium tartarate,calcium tartarate, lithium succinate, sodium succinate, potassiumsuccinate, calcium succinate, lithium acetate, sodium acetate, potassiumacetate, calcium acetate, sodium carbonate, potassium carbonate, calciumcarbonate, sodium bicarbonate, potassium bicarbonate, calciumbicarbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide,or combinations thereof. In some embodiments, the base includes acitrate. The citrate can include at least one of lithium citratemonohydrate, sodium citrate monohydrate, potassium citrate monohydrate,calcium citrate monohydrate, lithium citrate dihydrate, sodium citratedihydrate, potassium citrate dihydrate, calcium citrate dihydrate,lithium citrate trihydrate, sodium citrate trihydrate, potassium citratetrihydrate, calcium citrate trihydrate, lithium citrate tetrahydrate,sodium citrate tetrahydrate, potassium citrate tetrahydrate, calciumcitrate tetrahydrate, lithium citrate pentahydrate, sodium citratepentahydrate, potassium citrate pentahydrate, calcium citratepentahydrate, lithium citrate hexahydrate, sodium citrate hexahydrate,potassium citrate hexahydrate, calcium citrate hexahydrate, lithiumcitrate heptahydrate, sodium citrate heptahydrate, potassium citrateheptahydrate, or calcium citrate heptahydrate. In some embodiments, theliquid formulation includes at least one of sodium citrate monohydrate,potassium citrate monohydrate, calcium citrate monohydrate, sodiumcitrate dihydrate, potassium citrate dihydrate, calcium citratedihydrate, sodium citrate trihydrate, potassium citrate trihydrate,calcium citrate trihydrate, sodium citrate tetrahydrate, potassiumcitrate tetrahydrate, calcium citrate tetrahydrate, sodium citratepentahydrate, potassium citrate pentahydrate, calcium citratepentahydrate, sodium citrate hexahydrate, potassium citrate hexahydrate,calcium citrate hexahydrate, sodium citrate heptahydrate, potassiumcitrate heptahydrate, or calcium citrate heptahydrate.

In some embodiments, the base includes sodium citrate dihydrate.

In some embodiments, the formulation includes about 0.1 wt. % to about 5wt. % of a base such as citrate (e.g., sodium citrate dihydrate).

The liquid formulation can further include a sweetener. In someembodiments, the sweetener includes a sugar. The sugar can includesucrose. In some embodiments, the sweetener includes an intensesweetener. The intense sweetener can include sucralose.

In some embodiments, the sweetener is present in the liquid formulationin an amount of about 30 wt. % to about 70 wt. %. For example, thesweetener can be present in the liquid formulation in an amount of about45 wt. % to about 55 wt. %.

The liquid formulation can further include a bitterness masking agent.In some embodiments, the bitterness masking agent is present in theliquid formulation in an amount of about 0.01 wt. % to about 2 wt. %.For example, the bitterness masking agent can be present in the liquidformulation in an amount of about 0.2 wt. % to about 0.5 wt. %.

The liquid formulation can further include a flavoring agent. Theflavoring agent can include at least one of a natural flavoring agent, anatural fruit flavoring agent, an artificial flavoring agent, anartificial fruit flavoring agent, or a flavor enhancer. In someembodiments, the flavoring agent is present in the liquid formulation inan amount of about 0.01 wt. % to about 2 wt. %. For example, theflavoring agent can be present in the liquid formulation in an amount ofabout 0.01 wt. % to about 0.1 wt. %.

In some embodiments, the liquid formulation further includes a coloringagent.

In some embodiments, the liquid formulation is prepared from apharmaceutically acceptable salt of the compound of formula (I). Forexample, the liquid formulation can be prepared from the hydrogensulfate salt of the compound of formula (I).

A liquid formulation as provided herein can be prepared from apharmaceutically acceptable salt of the compound of formula (I) such asthe hydrogen sulfate salt. In some embodiments, the liquid formulationis prepared from a crystalline form of the compound of formula (I). Forexample, the crystalline form can have the formula (I-HS):

In some embodiments, the crystalline form is characterized by havingXRPD diffraction peaks (2θ degrees) at 18.4±0.2, 20.7±0.2, 23.1±0.2, and24.0±0.2. In some embodiments, the crystalline form is characterized byhaving XRPD diffraction peaks (2θ degrees) at 10.7±0.2, 18.4±0.2,20.7±0.2, 23.1±0.2, and 24.0±0.2. In some embodiments, the crystallineform is characterized by having XRPD diffraction peaks (2θ degrees) at10.7±0.2, 18.4±0.2, 19.2±0.2, 20.2±0.2, 20.7±0.2, 21.5±0.2, 23.1±0.2,and 24.0±0.2. In some embodiments, the crystalline form is characterizedby having XRPD diffraction peaks (2θ degrees) at 10.7±0.2, 15.3±0.2,16.5±0.2, 18.4±0.2, 19.2±0.2, 19.9±0.2, 20.2±0.2, 20.7±0.2, 21.5±0.2,22.1±0.2, 23.1±0.2, 24.0±0.2, 24.4±0.2, 25.6±0.2, 26.5±0.2, 27.6±0.2,28.2±0.2, 28.7±0.2, 30.8±0.2, and 38.5±0.2.

In some embodiments, the liquid formulation comprises a compound offormula (I), a pharmaceutically acceptable salt thereof, or acombination thereof;

a solubilizing agent; and

a buffer;

wherein:

-   -   the formulation has a pH of about 2.5 to about 5.5; and    -   the compound of formula (I) has a concentration of about 15        mg/mL to about 35 mg/mL in the liquid formulation.

In some embodiments, the formulation has a pH of about 3 to about 4. Insome embodiments, the buffer comprises sodium citrate dihydrate.

In some embodiments, the liquid formulation comprises a compound offormula (I), a pharmaceutically acceptable salt thereof, or acombination thereof;

a solubilizing agent; and

a base;

wherein:

-   -   the formulation has a pH of about 2.5 to about 5.5; and    -   the compound of formula (I) has a concentration of about 15        mg/mL to about 35 mg/mL in the liquid formulation.

In some embodiments, the formulation has a pH of about 3 to about 4. Insome embodiments, the base comprises sodium citrate dihydrate.

In some embodiments, the liquid formulation comprises a compound offormula (I), a pharmaceutically acceptable salt thereof, or acombination thereof;

a solubilizing agent;

a buffer;

a sweetener;

a bitterness masking agent; and

a flavoring agent,

wherein:

-   -   the formulation has a pH of about 3 to about 4; and    -   the compound of formula (I) has a concentration of about 15        mg/mL to about 35 mg/mL in the liquid formulation. In some        embodiments, the buffer comprises sodium citrate dihydrate. In        some embodiments, the sweetener comprises sucrose.

In some embodiments, the liquid formulation comprises a compound offormula (I), a pharmaceutically acceptable salt thereof, or acombination thereof;

a solubilizing agent;

a base;

a sweetener;

a bitterness masking agent; and

a flavoring agent,

wherein:

-   -   the formulation has a pH of about 3 to about 4; and    -   the compound of formula (I) has a concentration of about 15        mg/mL to about 35 mg/mL in the liquid formulation. In some        embodiments, the base comprises sodium citrate dihydrate. In        some embodiments, the sweetener comprises sucrose.

In some embodiments, the liquid formulation comprises a compound offormula (I), a pharmaceutically acceptable salt thereof, or acombination thereof;

a solubilizing agent present in an amount of about 5 wt. % to about 35wt. %;

a buffer present in an amount of about 0.1 wt. % to about 5 wt. %;

wherein:

-   -   the formulation has a pH of about 2.5 to about 5.5; and    -   the compound of formula (I) has a concentration of about 20        mg/mL to about 30 mg/mL in the liquid formulation.

In some embodiments, the liquid formulation comprises a compound offormula (I), a pharmaceutically acceptable salt thereof, or acombination thereof;

a solubilizing agent present in an amount of about 5 wt. % to about 35wt. %;

a base present in an amount of about 0.1 wt. % to about 5 wt. %;

wherein:

-   -   the formulation has a pH of about 2.5 to about 5.5; and    -   the compound of formula (I) has a concentration of about 20        mg/mL to about 30 mg/mL in the liquid formulation.

In some embodiments, the liquid formulation comprises a compound offormula (I), a pharmaceutically acceptable salt thereof, or acombination thereof;

a solubilizing agent present in an amount of about 5 wt. % to about 35wt. %;

a buffer present in an amount of about 0.1 wt. % to about 5 wt. %;

a sweetener present in an amount of about 30 wt. % to about 70 wt. %;

a bitterness masking agent present in an amount of about 0.2 wt. % toabout 0.5 wt. %; and

a flavoring agent present in an amount of about 0.01 wt. % to about 2wt. %,

wherein:

-   -   the formulation has a pH of about 2.5 to about 5.5; and    -   the compound of formula (I) has a concentration of about 20        mg/mL to about 30 mg/mL in the liquid formulation.

In some embodiments, the liquid formulation comprises a compound offormula (I), a pharmaceutically acceptable salt thereof, or acombination thereof;

a solubilizing agent present in an amount of about 5 wt. % to about 35wt. %;

a base present in an amount of about 0.1 wt. % to about 5 wt. %;

a sweetener present in an amount of about 30 wt. % to about 70 wt. %;

a bitterness masking agent present in an amount of about 0.2 wt. % toabout 0.5 wt. %; and

a flavoring agent present in an amount of about 0.01 wt. % to about 2wt. %,

wherein:

-   -   the formulation has a pH of about 2.5 to about 5.5; and    -   the compound of formula (I) has a concentration of about 20        mg/mL to about 30 mg/mL in the liquid formulation.

In some embodiments, the liquid formulation comprises a compound offormula (I), a pharmaceutically acceptable salt thereof, or acombination thereof;

a solubilizing agent present in an amount of about 5 wt. % to about 35wt. %;

a buffer comprising sodium citrate dihydrate present in an amount ofabout 0.1 wt. % to about 5 wt. %;

a sweetener comprising sucrose present in an amount of about 30 wt. % toabout 70 wt. %;

a bitterness masking agent is present in an amount of about 0.2 wt. % toabout 0.5 wt. %; and

a flavoring agent present in an amount of about 0.01 wt. % to about 2wt. %,

wherein:

-   -   the formulation has a pH of about 3 to about 4; and    -   the compound of formula (I) has a concentration of about 20        mg/mL to about 30 mg/mL in the liquid formulation.

In some embodiments, the liquid formulation comprises a compound offormula (I), a pharmaceutically acceptable salt thereof, or acombination thereof;

a solubilizing agent present in an amount of about 5 wt. % to about 35wt. %;

a base comprising sodium citrate dihydrate present in an amount of about0.1 wt. % to about 5 wt. %;

a sweetener comprising sucrose present in an amount of about 30 wt. % toabout 70 wt. %;

a bitterness masking agent is present in an amount of about 0.2 wt. % toabout 0.5 wt. %; and

a flavoring agent present in an amount of about 0.01 wt. % to about 2wt. %,

wherein:

-   -   the formulation has a pH of about 3 to about 4; and    -   the compound of formula (I) has a concentration of about 20        mg/mL to about 30 mg/mL in the liquid formulation.

In some embodiments, the liquid formulation comprises a compound offormula (I), a pharmaceutically acceptable salt thereof, or acombination thereof;

a cyclodextrin present in an amount of about 5 wt. % to about 35 wt. %;

sodium citrate dihydrate present in an amount of about 0.1 wt. % toabout 5 wt. %;

a sweetener comprising sucrose or a high intensity sweetener present inan amount of about 30 wt. % to about 70 wt. %;

a bitterness masking agent is present in an amount of about 0.2 wt. % toabout 0.5 wt. %; and

a flavoring agent present in an amount of about 0.01 wt. % to about 2wt. %,

wherein:

-   -   the formulation has a pH of about 3 to about 4; and    -   the compound of formula (I) has a concentration of about 20        mg/mL to about 30 mg/mL in the liquid formulation.

In some embodiments, the liquid formulation is an oral liquidformulation.

In some embodiments, the compound of formula (I), a pharmaceuticallyacceptable salt thereof, or a combination thereof, is administered in28-day cycles. In some embodiments, the compound is administered in adosage calculated to be equal to the exposure of an adult taking thecompound of formula (I), a pharmaceutically acceptable salt thereof, ora combination thereof, at a dose of 100 mg twice a day.

The features and advantages described in this summary and the followingdetailed description are not all-inclusive. Many additional features andadvantages will be apparent to one of ordinary skill in the art in viewof the drawings, specification, and claims hereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an X-ray powder diffraction (XRPD) pattern ofcrystalline form (I-HS) prepared according to Example 2.

FIG. 2 illustrates a simultaneous thermogravimetric/differential thermalanalyzer (TG/DTA) profile of crystalline form (I-HS) prepared accordingto Example 2.

FIG. 3 illustrates a differential scanning calorimetry (DSC) profile ofcrystalline form (I-HS) prepared according to Example 2.

FIGS. 4A and 4B illustrate polarized light microscopy (PLM) images ofcrystalline form (I-HS) prepared according to Example 2 under (A)unpolarized and (B) polarized light.

FIG. 5 illustrates a dynamic vapor sorption (DVS) isotherm profile ofcrystalline form (I-HS) prepared according to Example 2.

FIG. 6 illustrates an infrared (IR) spectroscopy profile of crystallineform (I-HS) prepared according to Example 2.

FIG. 7 illustrates an XRPD pattern of the amorphous freebase form of acompound of formula (I).

FIG. 8 illustrates an X-ray powder diffraction (XRPD) pattern ofcrystalline form (I-HS).

FIG. 9 is pictogram of pediatric solution formulation compoundinginstructions for the crystalline form (I-HS).

FIG. 10 is set of six MR images showing the brain in neck of the patientdiagnosed with infantile fibrosarcoma. (A) and (B) are MR images of thebrain and neck showing a 20 mm×19 mm×18 mm hyperenhancing mass involvingthe skull base of the middle cranial fossa, just anterior and inferiorto the inner ear structures five weeks following surgical resection. (C)and (D) are MR images of the brain and neck showing a significantinterval reduction in the size and enhancement of the mass by more than90% from baseline at the end of cycle 1 (day 28) where the patient wasadministered the hydrogen sulfate salt of(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamideBID. (E) and (F) are MR images of the brain and neck taken at the end ofCycle 2, which confirmed the size reduction and showed continueddecrease in enhancement, confirming partial response.

FIG. 11 is a sequence listing for an exemplary wildtype TrkA polypeptide(SEQ ID NO: 1).

FIG. 12 is a sequence listing for an exemplary wildtype TrkA polypeptide(SEQ ID NO: 2).

FIG. 13 is a sequence listing for an exemplary wildtype TrkA polypeptide(SEQ ID NO: 3).

DETAILED DESCRIPTION

The present disclosure relates to methods of treating pediatric cancers.The methods include administering(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide(formula (I)), pharmaceutically acceptable salts thereof, for examplethe hydrogen sulfate salt, a crystalline form of the hydrogen sulfatesalt, or pharmaceutical compositions including the compound of formula(I), for example liquid formulations including the compound of formula(I).

Methods of Treating a Pediatric Cancer

The present disclosure relates to methods of treating a pediatric cancerin a subject in need thereof. The methods include administering to thesubject a therapeutically effective amount of(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehaving the formula (I):

a pharmaceutically acceptable salt thereof, or a combination thereof.

In some embodiments, a patient is a pediatric patient (i.e., a patientunder the age of 21 years at the time of diagnosis or treatment). Theterm “pediatric” can be further divided into various subpopulationsincluding: neonates (from birth through the first 28 days of life);infants (29 days of age to less than two years of age); children (twoyears of age to less than 12 years of age); and adolescents (12 years ofage through 21 years of age (up to, but not including, the twenty-secondbirthday)). See, e.g., Berhman R E, Kliegman R, Arvin A M, Nelson W E.Nelson Textbook of Pediatrics, 15th Ed. Philadelphia: W.B. SaundersCompany, 1996; Rudolph A M, et al. Rudolph's Pediatrics, 21st Ed. NewYork: McGraw-Hill, 2002; and Avery M D, First L R. Pediatric Medicine,2nd Ed. Baltimore: Williams & Wilkins; 1994.

In some embodiments, the patient is from birth through the first 28 daysof life, from 29 days of age to less than two years of age, from twoyears of age to less than 12 years of age, or 12 years of age through 21years of age (up to, but not including, the twenty-second birthday). Insome embodiments, the patient is from birth through the first 28 days oflife, from 29 days of age to less than 1 year of age, from one month ofage to less than four months of age, from three months of age to lessthan seven months of age, from six months of age to less than 1 year ofage, from 1 year of age to less than 2 years of age, from 2 years of ageto less than 3 years of age, from 2 years of age to less than sevenyears of age, from 3 years of age to less than 5 years of age, from 5years of age to less than 10 years of age, from 6 years of age to lessthan 13 years of age, from 10 years of age to less than 15 years of age,or from 15 years of age to less than 22 years of age.

In some embodiments, the method further includes performing amorphological diagnosis prior to administering the compound of formula(I), or a pharmaceutically acceptable salt thereof, or a combinationthereof. The method can further include performing molecular testingprior to administering the compound of formula (I), or apharmaceutically acceptable salt thereof, or a combination thereof. Insome embodiments, the method includes performing morphological diagnosisand molecular testing prior to administering the compound of formula(I), or a pharmaceutically acceptable salt thereof, or a combinationthereof.

Some embodiments include the use of the compound of formula (I), apharmaceutically acceptable salt thereof, or a combination thereof forthe treatment of disorders and diseases in a pediatric subject, whichcan be treated by inhibiting TrkA, TrkB and/or TrkC kinases, such as aTrkA, TrkB and/or TrkC mediated condition, such as one or moreconditions described herein, including a Trk-associated cancer, in,e.g., an infant, child, or adolescent. The present disclosure is furtherdirected to pharmaceutical compositions comprising compound of formula(I), a pharmaceutically acceptable salt thereof, or a combinationthereof. In some embodiments, the pharmaceutical composition comprises acompound of formula (I), a pharmaceutically acceptable salt thereof, ora combination thereof and a pharmaceutically acceptable diluent orcarrier.

In some embodiments, provided herein is a method for treating apediatric patient diagnosed with a TRK-associated cancer, comprisingadministering to the pediatric patient a therapeutically effectiveamount of the compound of formula (I), a pharmaceutically acceptablesalt thereof, or a combination thereof.

The Trk family of neurotrophin receptors, TrkA, TrkB, and TrkC (encodedby the NTRK1, NTRK2, and NTRK3 genes, respectively) and theirneurotrophin ligands regulate growth, differentiation and survival ofneurons. Dysregulation in a NTRK gene, a Trk protein, or expression oractivity, or level of the same, such as translocations involving theNTRK kinase domain, mutations involving the TRK ligand-binding site,amplifications of a NTRK gene, Trk mRNA splice variants, andoverexpression of a NTRK gene (e.g., caused by Trk autocrine/paracrinesignaling) are described in a diverse number of tumor types and maycontribute to tumorigenesis. Translocations in NTRK1, NTRK2, and NTRK3that lead to the production of constitutively-active TrkA, TrkB, andTrkC fusion proteins are oncogenic and prevalent in a wide array oftumor types.

In some embodiments, the dysregulation in a NTRK gene, a Trk protein, orexpression or activity, or level of the same, includes overexpression ofa wild-type NTRK1, NTRK2, or NTRK3 gene (e.g., leading to autocrineactivation). In some embodiments, the dysregulation in a NTRK gene, aTrk protein, or expression or activity, or level of the same, includesoverexpression, activation, amplification, or mutation in a chromosomalsegment comprising the NTRK1, NTRK2, or NTKR3 gene or a portion thereof.In some embodiments, the dysregulation of a NTRK gene, a Trk protein, orexpression or activity, or level of the same, includes one or morechromosome translocations or inversions resulting in NTRK1, NTRK2, orNTRK3 gene fusions, respectively. In some embodiments, the dysregulationof a NTRK gene, a Trk protein, or expression or activity, or level ofthe same, is a result of genetic translocations in which the expressedprotein is a fusion protein containing residues from a non-TrkA partnerprotein and TrkA, a non-TrkB partner protein and TrkB, or a non-TrkCpartner protein and TrkC proteins, and include a minimum of a functionalTrkA, TrkB, or TrkC kinase domain, respectively.

In some embodiments, a TrkA fusion protein is one of the TrkA fusionproteins shown in Table 10. Additional rearrangements of NTRK weredetected in pediatric patients having papillary thyroid carcinomas(Sassolas et al., Thyroid 22:17-26, 2012).

TABLE 10 Exemplary Trk Fusion Proteins and Cancers in Pediatric SubjectsFusion Non-Trk Fusion Protein Partner Pediatric Cancer References ETV6-ETS variant gene Pediatric nephroma; Bouhana et al., AACR 103^(rd)Annual NTRK3 6, aka TEL Congenital Meeting, 2012, Abstract No. 1798;fibrosarcoma (CFS); Bourgeois et al., Am. J. Surg. Pathol. Pediatrichigh-grade 24: 937-946, 2000; glioma (HGG); Wu et al., Nat. Genet. 46:444-450, Mesenchymal cancers 2014; (infant fibrosarcoma Tognon et al.,Cancer Cell 2: 367-376, (IF); 2002; Congenital Euhus et al., Cancer Cell2: 347-348, mesoblastic 2002; nephroma; Sheng et al., Am. J. Clin.Pathol. Congenital infantile 115: 348-355, 2001; fibrosarcoma (CIFS);Jones et al., Nat. Genet 45: 927-932, Pilocytic astrocytoma; 2013; braintumors Carvalho et al., Neuro-Oncology (glioglastomas); 17: iii1-iii40,2015, Abstract No. HG- Pediatic acute 09. leukemia; Shah et al.,Pediatr. Blood Cancer Ph-like Acute 59: 179-181, 2012; LymphoblasticEguchi et al., Med. Pediatr. Oncol. Leukemia; 37: 417, 2001; CellularCongenital Prasad et al., Cancer 122: 1097-1017, Mesoblastic 2016.Nephroma (CMN); Roberts et al., N. Engl. J. Med. Infantile fibrosarcoma;371: 1005-1015, 2014, ALK-negative Alassiri et al., Am J Surg Pathol.,2016 inflammatory August; 40(8): 1051-61, 2016. myofibroblasticNagasubramanian et al., Pediatr Blood tumors (IMT); Cancer., Aug; 63(8):1468-70, 2016. Mammary Carcinoma Hyrcza et al., Vol. 469, Supp. (e.g.,Mammary Supplement 1, pp. S17. Abstract Analogue Secretory Number:OFP-1997-7; 31st Carcinoma, Secretory International Congress of theBreast Carcinoma) International Academy of Pathology and the 28thCongress of the European Society of Pathology, Cologne, Germany. 25-29Sep. 2016. RET/ RET Papillary Thyroid Bongarzone et al., J. Clin.Endocrinol. NTRK1 Carcinomas Metab. 81: 2006-2009, 1996. TPM3- TPM3-actin Pediatric high-grade Wu et al., Nat. Genet. 46: 444-450, NTRK1bingind protein glioma (HGG), diffuse 2014; intrinsic pontine Drexler etal., Leukemia 14: 1533-1559, gliomas (DIPGs) and 2000; non-brainstemHGGs Jones et al., Nat. Genet. 45: 927-932, (NBS-HGGs); 2013; Anaplasticlarge cell Beimfohr et al., Int. J. Cancer 80: 842- lymphoma (ALCL) 847,1999; and non-Hodgkin's US 2016/0009785; lymphoma (NHL); Doebele et al.,Cancer Discov. 5: 1049- Pilocytic astrocytoma; 1057, 2015; pediatricpapillary Wu et al., Modern Pathol. 29: 359-369, thyroid carcinoma,2016. soft tissue sarcoma; spitzoid melanomal BTBD1- TopoisomerasePediatric high-grade Wu et al., Nat. Genet. 46: 444-450, NTRK3I-interacting glioma (HGG), diffuse 2014. protein intrinsic pontinegliomas (DIPGs) and non-brainstem HGGs (NBS-HGGs) VCL- Actin-bindingPediatric high-grade Wu et al., Nat. Genet. 46: 444-450, NTRK2 proteinvinculin glioma (HGG), diffuse 2014. intrinsic pontine gliomas (DIPGs)and non-brainstem HGGs (NBS-HGGs) AGBL4- ATP/GTP Pediatric high-grade Wuet al., Nat. Genet. 46: 444-450, NTRK2 binding protein glioma (HGG),diffuse 2014. intrinsic pontine gliomas (DIPGs) and non-brainstem HGGs(NBS-HGGs) LMNA- Lamin A/C Congenital infantile Wong et al., J. Nat.Cancer Inst. NTRK1 fibrosarcoma (CIFS); 108(1), 2016; soft-tissuesarcoma, Doebele et al., Cancer Discov. 5: 1049- Paediatric 1057, 2015;haemangiopericytoma- US 2016/0009785; like sarcoma; Haller et al., J.Pathol. 238: 700-710, Spindle cell sarcoma, 2016. NOS withmyo/haemangiopericytic growth pattern TFG- “Trk-fused gene” Anaplasticlarge cell Drexler et al., Leukemia 14: 1533-1559, NTRK1 lymphoma (ALCL)2000 and non-Hodgkin's lymphoma (NHL) QKI-NTRK2 KH domain Pilocyticastrocytoma Jones et al., Nat. Genet. 45: 927-932, containing RNA 2013,binding NACC2- NACC family Pilocytic astrocytoma Jones et al., Nat.Genet. 45: 927-932, NTRK2 member 2 2013. TPR- TPR Pediatic papillaryBeimfohr et al., Int. J. Cancer 80: 842- NTRK1 thyroid carcinoma 847,1999; Prasad et al., Cancer 122: 1097-1017, 2016. RABGAP1L- RABGAP1L US2016/0009785 NTRK1 MPRIP- MPRIP E.g., Lung cancer US 2016/0009785; NTRK1US 2015/0073036 SQSTM1- SQSTM1 Soft Tissue Sarcoma Doebele et al.,Cancer Discov. 5: 1049- NTRK1 1057, 2015 EML4- EML4 Advanced PediatricHarris et al., JAMA Oncol. Epub. Jan. NTRK3 Solid Tumors; 28, 2016.Pediatric Sims et al., Journal of Immunotherapy Fibrosarcoma of Cancer,Vol. 4, Supp. Supplement 1; Abstract Number: P280; 31st Annual Meetingand Associated Programs of the Society for Immunotherapy of Cancer, SITC2016. National Harbor, MD; 9-13 Nov. 2016. AFAP1- Actin FilamentPilocytic Astrocytoma Lin et al., Neuro-Oncol, Vol. 18, NTRK2 AssociatedWith Anaplasia Supp. Supplement 3, pp. iii58, Protein 1 Abstract Number:HG-48; 17^(th) International Symposium on Pediatric Neuro-Oncology,ISPNO 2016. Liverpool, UK, 12 Jun. 2016-15 Jun. 2016.

In some embodiments, the dysregulation of a NTRK gene, a Trk protein, orexpression or activity, or level of the same, includes one or moredeletions, insertions, or point mutation(s) in a Trk protein. In someembodiments, the dysregulation of a NTRK gene, a Trk protein, orexpression or activity, or level of the same, includes a deletion of oneor more residues from the TrkA protein, resulting in constitutiveactivity of the Trk kinase domain. In some embodiments, thedysregulation of a NTRK gene, a Trk protein, or expression or activity,or level of the same, includes at least one point mutation in a NTRK1gene that results in the production of a TrkA protein that has one ormore amino acid substitutions as compared to the wildtype TrkA protein(see, for example, the point mutations listed in Tables 11 and 12). Anexemplary wildtype TrkA polypeptide is SEQ ID NO: 1, an exemplarywildtype TrkB polypeptide is SEQ ID NO: 2, and an exemplary TrkCpolypeptide is SEQ ID NO: 3.

TABLE 11 Activating TrkA Point Mutations Mutation Pediatric CancerReference C6773T, C7232T, TrkA neuroblastoma Scaruffi et al., Int. J.C7301T Oncol. 14: 935-938, 1999

TABLE 12 Activating TrkA Point Mutations^(A) Exemplary Isoform in Pointwhich Mutation is Mutation Rationale Present (if known) R33W^(B)NP_001007793.1^(F) A336E Near NGF Binding Site Reference TrkA sequenceA337T Near NGF Binding Site Reference TrkA sequence R324Q or Near NGFBinding Site Unknown R324W V420M Close to Membrane Reference TrkAsequence R444Q or Close to Membrane Reference TrkA sequence R444W G517Ror P-Loop Reference TrkA sequence G517V K538A Activating Reference TrkAsequence V573M^(E) Reference TrkA sequence F589L^(E) Reference TrkAsequence G595R or Catalytic Domain Reference TrkA sequence G667C^(D)F598L^(E) Unknown R649W or Arginine may stabilize auto- Reference TrkAsequence R649L inhibited conformation. R682S Activation Loop ReferenceTrkA sequence V683G Activation Loop Reference TrkA sequence R702CExposed, may form face-to-face Reference TrkA sequence disulfide linkeddimer Q627X^(C), NP_001012331.1^(G), Q597X^(C), NP_001007793.1^(F), andQ633X^(C) Reference TrkA sequence, respectively ^(A)Reference TrkAsequence is UniProtKB/Swiss-Prot: P04629.4, and can be found at URL:www.ncbi.nlm.nih.gov/protein/94730402?report=genbank&log$=protalign&blast_rank=0&RID=0(SEQ ID NO. 1) ^(B)Zhang et al., Blood 124(21): 1682, 2014. Mutationfound in T-cell prolymphocytic leukemia. ^(C)Park et al., Proc. Natl.Acad. Sci. U.S.A. 112(40): 12492-12497, 2015. Mutation found incolorectal cancer. ^(D)Russo et al., Cancer Discov. Jan; 6(1): 36-44,2016. ^(E)PCT Application No. WO2016196141A1.^(F)www.ncbi.nlm.nih.gov/protein/56118210?report=genbank&log$=protalign&blast_rank=3&RID=0^(G)www.ncbi.nlm.nih.gov/protein/59889558

In some embodiments, the dysregulation of a NTRK gene, a Trk protein, orexpression or activity, or level of the same, includes a splicevariation in a TrkA mRNA which results in an expressed protein that isan alternatively spliced variant of TrkA having at least one residuedeleted (as compared to a wild-type TrkA protein) resulting inconstitutive activity of the TrkA kinase domain. In some embodiments, analternatively spliced form of TrkA with constitutive activity hasdeletions of exons 8, 9, and 11 resulting in an expressed proteinmissing residues 192-284 and 393-398 relative to TrkA Isoform 2, has adeletion of exon 10 in TrkA, or has a deletion in a NTRK1 gene thatencodes a TrkA protein with a 75 amino acid deletion in thetransmembrane domain (Reuther et al., Mol. Cell Biol. 20:8655-8666,2000).

Cancers identified as having dysregulation of a NTRK gene, a Trkprotein, or expression or activity, or level of the same, (seereferences cited herein and also the www.cancer.gov and www.nccn.orgwebsites) include:

(A) Cancers wherein the dysregulation of a NTRK gene, a Trk protein, orexpression or activity, or level of the same, includes one or morechromosome translocations or inversions resulting in TrkA fusionproteins, e.g., including:

Cancer Standard of Care Non-Small Cell radiotherapy (e.g., radioiodidetherapy, external-beam Lung Cancer² radiation, or radium 223 therapy),chemotherapeutics as single agents (e.g., afatinib dimaleate,bevacizumab, carboplatin, cetuximab, cisplatin, crizotinib, erlotinib,gefitinib, gemcitabine, methotrexate, paclitaxel, or pemetrexed) orcombinations (e.g., carboplatin- paclitaxel, gemcitabine-paclitaxel, orchemoradiation) Papillary Radiotherapies (e.g., radioiodide therapy orexternal- Thyroid beam radiation) and chemotherapeutics (e.g..sorafenib, Carcinoma¹⁴ sunitinib, or pazopanib) GlioblastomaChemotherapeutics (e.g., bevacizumab, everolimus, Multiforme¹⁵lomustine, or temozolomide) Colorectal Chemotherapeutics as singleagents (e.g., aflibercept, Carcinoma¹⁶ bevacizumab, capecitabine,cetuximab, fluorouracil, irinotecan, leucovorin, oxaliplatin,panitumumab, or regorafenib) or combinations (e.g., folfox, folfiri,capox, folfiri-bevacizumab, folfiri-cetuximab, or xelox) Melanoma¹²Chemotherapeutics (e.g., aldesleukin, dabrafenib, dacarbazine,interferon alfa-2b, ipilimumab, peginterferon alfa-2b, trametinib,orvemurafenib)

(B) Cancers wherein the dysregulation of a NTRK gene, a Trk protein, orexpression or activity, or level of the same, includes one or moredeletions, insertions, or mutations in the TrkA protein, e.g.,including:

Cancer Standard of care Acute Myeloid Chemotherapeutics as single agents(e.g., arsenic leukemia^(17,) ¹⁸ trioxide, cyclophosphamide, cytarabine,daunorubicin, doxorubicin, or vincristine) or combinations (e.g., ADE)Large Cell Radiotherapy (e.g., radioiodide therapy, external-Neuroendocrine beam radiation, or radium 223 therapy) and/or Carcinoma¹⁹chemotherapeutics (e.g., cisplatin, carboplatin, or etoposide)Neuroblastoma²⁰ Chemotherapeutics (e.g., cyclophosphamide, doxorubicin,or vincristine)

(C) Cancers wherein the dysregulation of a NTRK gene, a Trk protein, orexpression or activity, or level of the same, includes overexpression ofwildtype TrkA (autocrine activation), e.g., including:

Cancer Standard of care Prostate Radiotherapy (e.g., radium 223 therapy)or chemo- Carcinoma^(21, 22) therapeutics (e.g. abiraterone,cabazitaxel, degarelix, denosumab, docetaxel, enzalutamide, leuprolide,prednisone, or sipuleucel-T) Neuroblastoma²³ Chemotherapeutics (e.g.,cyclophosphamide, doxorubicin, or vincristine) PancreaticChemotherapeutics as single agents (e.g., erlotinib, Carcinoma²⁴fluorouracil, gemcitabine, or mitomycin C) or combi- nations (e.g.,gemcitabine-oxaliplatin) Melanoma²⁵ Chemotherapeutics (e.g.,aldesleukin, dabrafenib, dacarbazine, interferon alfa-2b, ipilimumab,peginterferon alfa-2b, trametinib, or vemurafenib) Head and NeckRadiotherapy and/or chemotherapeutics (e.g., Squamous Cell bleomycin,cetuximab, cisplatin, docetaxel, Carcinoma²⁶ fluorouracil, ormethotrexate) Gastric Chemotherapeutics (e.g., docetaxel, doxorubucin,Carcinoma²⁷ fluorouracil, mitomycin C, or trastuzumab)

In some embodiments, the dysregulation of a NTRK gene, a Trk protein, orexpression or activity, or level of the same, includes at least onepoint mutation in a NTRK1 gene that results in the production of a TrkBprotein that has one or more amino acid substitutions as compared to thewildtype TrkB protein (see, for example, the point mutations listed inTable 13.

TABLE 13 Activating TrkB Point Mutations^(A) Exemplary Isoform in whichPoint Mutation Rationale Mutation is Present (if known) A13T^(C)Reference TrkB sequence E142K^(C) Reference TrkB sequence R136H^(C)Reference TrkB sequence V619M^(B) Unknown F633L^(B) NP_006171.2^(D)(Corresponding to position 617 of Reference TrkB sequence) G639R^(B)NP_006171.2^(D) (Corresponding to position 623 of Reference TrkBsequence) G709C or G709A NP_006171.2^(D) (Corresponding or G709S^(B) toposition 693 of Reference TrkB sequence) ^(A)Reference TrkB sequence isUniProtKB/Swiss-Prot: Q16620.1, and can be found at URL:www.ncbi.nlm.nih.gov/protein/2497560?report=genbank&log$=protalign&blast_rank=0&RID=0)(SEQ ID NO. 2) ^(B)PCT Application No. WO2016196141A1. ^(C)Bonanno etal., Journal of Thoracic Oncology, Vol. 11, No. 4, Supp. Suppl. 1, ppS67. Abstract Number: 28P; 6^(th) European Lung Cancer Conference, ELCC2016, Geneva, Switzerland. ^(D)www.ncbi.nlm.nih.gov/protein/NP_006171.2

In some embodiments, the dysregulation of a NTRK gene, a Trk protein, orexpression or activity, or level of the same, includes at least onepoint mutation in a NTRK1 gene that results in the production of a TrkCprotein that has one or more amino acid substitutions as compared to thewildtype TrkC protein (see, for example, the point mutations listed inTable 14.

TABLE 14 Activating TrkC Point Mutations^(A) Exemplary Isoform in whichMutation is Present (if Point Mutation Rationale known) V603M^(C)NP_001007157.1^(D) F617L^(C) Reference TrkC sequence G623R^(B,C) StericHinderance Reference TrkC sequence G696C or G696A Reference TrkCsequence or G696S^(C) ^(A)Reference TrkC sequence isUniProtKB/Swiss-Prot: Q16288.2, and can be found at URL:www.ncbi.nlm.nih.gov/protein/134035335?report=genbank&log$=protalign&blast_rank=0&RID=0 (SEQ ID NO. 3) ^(B)Drilon et al., Ann Oncol. 2016 May; 27(5):920-6. doi: 10.1093/annonc/mdw042. Epub 2016 Feb. 15. ^(C)PCTApplication No. WO2016196141A1.^(D)www.ncbi.nlm.nih.gov/protein/NP_001007157In some embodiments, a TRK-associated cancer has been identified ashaving one or more TRK inhibitor resistance mutations (that result in anincreased resistance to a TRK inhibitor. Non-limiting examples of TRKinhibitor resistance mutations are listed in Tables 15-17.

TABLE 15 Exemplary TrkA Resistance Mutations Amino acid position 517(e.g., G517R) Amino acid position 542 (e.g., A542V) Amino acid position568 (e.g., Q568x) Amino acid position 573 (e.g., V573M) Amino acidposition 589 (e.g., F589L, F589C) Amino acid position 595 (e.g., G595S,G595R¹) Amino acid position 599 (e.g., D596V) Amino acid position 600(e.g., F600L) Amino acid position 602 (e.g., R602x) Amino acid position646 (e.g., F646V) Amino acid position 656 (e.g., C656Y, C656F) Aminoacid position 657 (e.g., L657V) Amino acid position 667 (e.g., G667C¹,G667S) Amino acid position 676 (e.g., Y676S) ¹Russo et al., AcquiredResistance to the TRK Inihibitor Entrectinib in Colorectal Cancer,Cancer Discov. January; 6(1):36-44, 2016.

TABLE 16 Exemplary TrkB Resistance Mutations Amino acid position 545(e.g., G545R) Amino acid position 570 (e.g., A570V) Amino acid position596 (e.g., Q596E, Q596P) Amino acid position 601 (e.g., V601G) Aminoacid position 617 (e.g., F617L, F617C, F617I) Amino acid position 623(e.g., G623S, G623R) Amino acid position 624 (e.g., D624V) Amino acidposition 628 (e.g., F628x) Amino acid position 630 (e.g., R630K) Aminoacid position 672 (e.g., F672x) Amino acid position 682 (e.g., C682Y,C682F) Amino acid position 683 (e.g., L683V) Amino acid position 693(e.g., G693S) Amino acid position 702 (e.g., Y702x)

TABLE 17 Exemplary TrkC Resistance Mutations Amino acid position 545(e.g., G545R) Amino acid position 570 (e.g., A570V) Amino acid position596 (e.g., Q596x) Amino acid position 601 (e.g., V601) Amino acidposition 617 (e.g., F617x, F617L) Amino acid position 623 (e.g., G623R¹)Amino acid position 624 (e.g., D624V) Amino acid position 628 (e.g.,F628x) Amino acid position 630 (e.g., R630x) Amino acid position 675(e.g., F675x) Amino acid position 685 (e.g., C685Y, C684F) Amino acidposition 686 (e.g., L686V) Amino acid position 696 (e.g., G696x, G696A)Amino acid position 705 (e.g., Y705x) ¹Drilon et al., What hides behindthe MASC: clinical response and acquired resistance to entrectinib afterETV6-NTRK3 identification in a mammary analogue secretory carcinoma(MASC), Ann Oncol. 2016 May; 27(5): 920-6. doi: 10.1093/annonc/mdw042.Epub 2016 Feb. 15.

In some embodiments, the dysregulation of a NTRK gene, a Trk protein, orexpression or activity, or level of the same, includes a splicevariation in a TrkA mRNA which results in an expressed protein that isan alternatively spliced variant of TrkA having at least one residuedeleted (as compared to a wild-type TrkA protein) resulting inconstitutive activity of the TrkA kinase domain. In some embodiments, analternatively spliced form of TrkA with constitutive activity is theTrkAIII splice variant and, e.g., is associated withneuroectodermal-derived tumors including Wilm's tumor, neuroblastoma,and medulloblastoma (see, e.g., U.S. Patent Publication No.2015/0218132).

Overexpression or increased expression of a Trk gene (e.g., as comparedto a control non-cancerous cell of the same cell type) is another typeof dysregulation of a NTRK gene that is associated with a variety ofdifferent pediatric cancers. For example, overexpression of a Trkreceptor has been observed in neuroectodermal-derived tumors includingWilm's tumor, neuroblastoma, and medulloblastoma (see, e.g., U.S. PatentApplication Publication No. 2015/0218132), overexpression of NTRK2 inpediatric colorectal cancer subjects indicates poor prognosis insubjects (see, e.g., Tanaka et al., PLoS One 9:E96410, 2014),overexpression of NTRK2 has been observed in medulloblastoma andneuroblastoma in pediatric subjects (see, e.g., Evans et al., Clin.Cancer Res. 5:3592-3602, 1999; Geiger et al., J. Cancer Res. 65:7033,2005). Decreased NTRK1 expression has been detected in bilateral stageIV adrenal neuroblastoma with multiple skin metastases in a neonate(see, e.g., Yanai et al., J. Pediatr. Surg. 39:1782-1783, 2004).

In some embodiments, a Trk-associated cancer is advanced solid andprimary central nervous system tumors (e.g., advanced solid and primarycentral nervous system tumors that are refractory to standard therapy).In some embodiments, the cancer is a solid or central nervous systemtumors (e.g., advanced solid or primary central nervous system tumor)that is refractory to standard therapy.

Cancers identified as having dysregulation of a NTRK gene, a Trkprotein, or expression or activity, or level of the same (see referencescited herein and also the www.cancer.gov and www.nccn.org websites)include:

(A) Cancers wherein the dysregulation of a NTRK gene, a Trk protein, orexpression or activity, or level of the same, includes one or morechromosome translocations or inversions resulting in Trk fusionproteins;

(B) Cancers wherein the dysregulation of a NTRK gene, a Trk protein, orexpression or activity, or level of the same, includes one or moredeletions, insertions, or mutations in the Trk protein;

(C) Cancers wherein the dysregulation of a NTRK gene, a Trk protein, orexpression or activity, or level of the same, includes overexpression ofwildtype Trk (e.g., leading to autocrine activation of a Trk);

In some embodiments, the dysregulation of a NTRK gene, a Trk protein, orexpression or activity, or level of the same, includes a translocationthat results in the expression of a TrkA, TrkB, or TrkC fusion protein,e.g., one of the TrkA, TrkB, or TrkC fusion proteins shown in Table 10.

In some embodiments, provided herein is a method for treating apediatric patient diagnosed with a Trk-associated cancer, comprisingadministering to the patient a therapeutically effective amount of thecompound of formula (I), a pharmaceutically acceptable salt thereof, ora combination thereof. For example, the Trk-associated cancer can beselected from the group consisting of: pediatric nephroma, congenitalfibrosarcoma (CFS), pediatric high-grade glioma (HGG), mesenchymalcancers (infant fibrosarcoma (IF), congenital mesoblastic nephroma,congenital infantile fibrosarcoma (CIFS); pilocytic astrocytoma, braintumors (e.g., glioglastomas), pediatic acute leukemia, Ph-like acutelymphoblastic leukemia, cellular congenital mesoblastic nephroma (CMN);infantile fibrosarcoma, pediatric high-grade glioma (HGG), diffuseintrinsic pontine gliomas (DIPGs), non-brainstem HGGs (NBS-HGGs),anaplastic large cell lymphoma (ALCL), non-Hodgkin's lymphoma (NHL),pediatric papillary thyroid carcinoma, secretory breast cancer, softtissue sarcoma, spitzoid melanoma, pediatric hemangiopericytoma-likesarcoma, spindle cell sarcoma, NOS with myo/haemangiopericytic growthpattern, advanced pediatric solid tumors, neuroectodermal-derived tumors(e.g., Wilm's tumor, neuroblastoma, and medulloblastoma), pediatriccolorectal cancer, adrenal neuroblastoma, and central nervous systemtumors (e.g., advanced solid and primary central nervous system tumorsthat are refractory to standard therapy).

The pediatric cancer can be a fibrosarcoma. For example, the pediatriccancer can be infantile fibrosarcoma. In some embodiments, the subjectis an infant and the fibrosarcoma is infantile fibrosarcoma.

In some embodiments, the pediatric cancer is amyofibroblastic/fibroblastic tumor. The pediatric cancer can be a solidtumor or a primary CNS tumor. The pediatric cancer can also be acongenital mesoblastic nephroma.

In some embodiments, a compound of formula (I), a pharmaceuticallyacceptable salt thereof, or a combination thereof is useful for treatingTrk-associated cancers in pediatric patients. For example, the compoundsprovided herein can be used to treat infantile sarcoma, pediatricglioma, neuroblastoma, congenital mesoblastic nephroma, brain low-gradeglioma, and pontine glioma.

In some embodiments, the Trk-associated cancer is a glioma. For example,the Trk-associated cancer is selected from the group consisting of:pediatric high-grade glioma (HGG), diffuse intrinsic pontine gliomas(DIPGs), and on-brainstem HGGs (NBS-HGGs).

In some embodiments, the pediatric cancer is an extracranial solidtumor. For example, the pediatric cancer is selected from the groupconsisting of: neuroblastoma, nephroblastoma (e.g., Wilm's tumor),rhabdomyosarcoma and hepatoblastoma

In some embodiments, a compound of formula (I), a pharmaceuticallyacceptable salt thereof, or a combination thereof is useful for treatinga Trk-associated cancer in combination with one or more additionaltherapeutic agents or therapies that work by the same or a differentmechanism of action.

In some embodiments, the additional therapeutic agent(s) is selectedfrom the group of: receptor tyrosine kinase-targeted therapeutic agents,including cabozantinib, crizotinib, erlotinib, gefitinib, imatinib,lapatinib, nilotinib, pazopanib, pertuzumab, regorafenib, sunitinib, andtrastuzumab.

In some embodiments, the additional therapeutic agent(s) is selectedfrom signal transduction pathway inhibitors, including, e.g.,Ras-Raf-MEK-ERK pathway inhibitors (e.g., sorafenib, trametinib, orvemurafenib), PI3K-Akt-mTOR-S6K pathway inhibitors (e.g., everolimus,rapamycin, perifosine, or temsirolimus) and modulators of the apoptosispathway (e.g., obataclax).

In some embodiments, the additional therapeutic agent(s) is selectedfrom the group of: cytotoxic chemotherapeutics, including, e.g., arsenictrioxide, bleomycin, cabazitaxel, capecitabine, carboplatin, cisplatin,cyclophosphamide, cytarabine, dacarbazine, daunorubicin, docetaxel,doxorubicin, etoposide, fluorouracil, gemcitabine, irinotecan,lomustine, methotrexate, mitomycin C, oxaliplatin, paclitaxel,pemetrexed, temozolomide, and vincristine.

In some embodiments, the additional therapeutic agent(s) is selectedfrom the group of angiogenesis-targeted therapies, including e.g.,aflibercept and bevacizumab.

In some embodiments, the additional therapeutic agent(s) is selectedfrom the group of immune-targeted agents, e.g., including aldesleukin,ipilimumab, lambrolizumab, nivolumab, and sipuleucel-T.

In some embodiments, the additional therapeutic agent(s) is selectedfrom agents active against the downstream Trk pathway, including, e.g.,NGF-targeted biopharmaceuticals, such as NGF antibodies and panTrkinhibitors.

In some embodiments, the additional therapeutic agent or therapy isradiotherapy, including, e.g., radioiodide therapy, external-beamradiation, and radium 223 therapy.

In some embodiments, the additional therapeutic agent(s) includes anyone of the above listed therapies or therapeutic agents which arestandards of care in cancers wherein the cancer has a dysregulation of aNTRK gene, a Trk protein, or expression or activity, or level of thesame.

Methods of detecting dysregulation of a NTRK gene, a Trk protein, orexpression or activity, or level of the same, include, e.g., detectionof NTRK gene translocations, e.g., using Fluorescent In SituHybridization (FISH) (e.g., as described in International ApplicationNos. PCT/US2013/061211 and PCT/US2013/057495, which are incorporatedherein by reference).

In some embodiments, provided herein is a method of treating cancer(e.g., a Trk-associated cancer) in a pediatric patient, comprisingadministering to said patient compound of formula (I), apharmaceutically acceptable salt thereof, or a combination thereof incombination with at least one additional therapy or therapeutic agent.In some embodiments, the at least one additional therapy or therapeuticagent is selected from radiotherapy (e.g., radioiodide therapy,external-beam radiation, or radium 223 therapy), cytotoxicchemotherapeutics (e.g., arsenic trioxide, bleomycin, cabazitaxel,capecitabine, carboplatin, cisplatin, cyclophosphamide, cytarabine,dacarbazine, daunorubicin, docetaxel, doxorubicin, etoposide,fluorouracil, gemcitabine, irinotecan, lomustine, methotrexate,mitomycin C, oxaliplatin, paclitaxel, pemetrexed, temozolomide, orvincristine), tyrosine kinase targeted-therapeutics (e.g., afatinib,cabozantinib, cetuximab, crizotinib, dabrafenib, erlotinib, gefitinib,imatinib, lapatinib, nilotinib, pazopanib, panitumumab, pertuzumab,regorafenib, sunitinib, or trastuzumab), apoptosis modulators and signaltransduction inhibitors (e.g. everolimus, perifosine, rapamycin,sorafenib, temsirolimus, trametinib, or vemurafenib), immune-targetedtherapies (e.g., aldesleukin, interferon alfa-2b, ipilimumab,lambrolizumab, nivolumab, prednisone, or sipuleucel-T) andangiogenesis-targeted therapies (e.g., aflibercept or bevacizumab),wherein the amount of a compound provided herein or a pharmaceuticallyacceptable salt thereof is, in combination with the additional therapyor therapeutic agent, effective in treating the cancer.

In some embodiments, the additional therapeutic agent is a different Trkinhibitor. In some embodiments, a receptor tyrosine kinase targetedtherapeutic is a multikinase inhibitor (e.g., TRK-targeted therapeuticinhibitor) exhibiting TRK inhibition activity. In some embodiments, theTRK-targeted therapeutic inhibitor is selective for a TRK kinase.Exemplary TRK kinase inhibitors can exhibit inhibition activity (IC₅₀)against a TRK kinase of less than about 1000 nM, less than about 500 nM,less than about 200 nM, less than about 100 nM, less than about 50 nM,less than about 25 nM, less than about 10 nM, or less than about 1 nM asmeasured in an assay as described herein. In some embodiments, a TRKkinase inhibitor can exhibit inhibition activity (IC₅₀) against a TRKkinase of less than about 25 nM, less than about 10 nM, less than about5 nM, or less than about 1 nM as measured in an assay. For example, aTRK inhibitor assay can be any of those provided in U.S. Pat. No.8,933,084 (e.g., Example A or B).

Non-limiting examples of receptor tyrosine kinase (e.g., Trk) targetedtherapeutic agents, include afatinib, cabozantinib, cetuximab,crizotinib, dabrafenib, entrectinib, erlotinib, gefitinib, imatinib,lapatinib, lestaurtinib, nilotinib, pazopanib, panitumumab, pertuzumab,sunitinib, trastuzumab,1-((3S,4R)-4-(3-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-3-(2-methylpyrimidin-5-yl)-1-phenyl-1H-pyrazol-5-yl)urea,AG 879, AR-772, AR-786, AR-256, AR-618, AZ-23, AZ623, DS-6051, Gö 6976,GNF-5837, GTx-186, GW 441756, LOXO-101, MGCD516, PLX7486, RXDX101,TPX-0005, and TSR-011. Additional Trk targeted therapeutic agentsinclude those described in U.S. Pat. Nos. 8,450,322; 8,513,263;8,933,084; 8,791,123; 8,946,226; 8,450,322; 8,299,057; and 8,912,194;U.S. Publication No. 2016/0137654; 2015/0166564; 2015/0051222;2015/0283132; and 2015/0306086; International Publication No. WO2010/033941; WO 2010/048314; WO 2016/077841; WO 2011/146336; WO2011/006074; WO 2010/033941; WO 2012/158413; WO 2014078454; WO2014078417; WO 2014078408; WO 2014078378; WO 2014078372; WO 2014078331;WO 2014078328; WO 2014078325; WO 2014078323; WO 2014078322; WO2015175788; WO 2009/013126; WO 2013/174876; WO 2015/124697; WO2010/058006; WO 2015/017533; WO 2015/112806; WO 2013/183578; and WO2013/074518, all of which are hereby incorporated by reference in theirentireties.

Further examples of Trk inhibitors can be found in U.S. Pat. No.8,637,516, International Publication No. WO 2012/034091, U.S. Pat. No.9,102,671, International Publication No. WO 2012/116217, U.S.Publication No. 2010/0297115, International Publication No. WO2009/053442, U.S. Pat. No. 8,642,035, International Publication No. WO2009092049, U.S. Pat. No. 8,691,221, International Publication No.WO2006131952, all of which are incorporated by reference in theirentireties herein. Exemplary Trk inhibitors include GNF-4256, describedin Cancer Chemother. Pharmacol. 75(1):131-141, 2015; and GNF-5837(N-[3-[[2,3-dihydro-2-oxo-3-(1H-pyrrol-2-ylmethylene)-1H-indol-6-yl]amino]-4-methylphenyl]-N′-[2-fluoro-5-(trifluoromethyl)phenyl]-urea),described in ACS Med. Chem. Lett. 3(2): 140-145, 2012, each of which isincorporated by reference in its entirety herein. Additional examples ofTrk inhibitors include those disclosed in U.S. Publication No.2010/0152219, U.S. Pat. No. 8,114,989, and International Publication No.WO 2006/123113, all of which are incorporated by reference in theirentireties herein. Exemplary Trk inhibitors include AZ623, described inCancer 117(6):1321-1391, 2011; AZD6918, described in Cancer Biol. Ther.16(3):477-483, 2015; AZ64, described in Cancer Chemother. Pharmacol.70:477-486, 2012; AZ-23((S)-5-Chloro-N2-(1-(5-fluoropyridin-2-yl)ethyl)-N4-(5-isopropoxy-1H-pyrazol-3-yl)pyrimidine-2,4-diamine),described in Mol. Cancer Ther. 8:1818-1827, 2009; and AZD7451; each ofwhich is incorporated by reference in its entirety.

A Trk inhibitor can include those described in U.S. Pat. Nos. 7,615,383;7,384,632; 6,153,189; 6,027,927; 6,025,166; 5,910,574; 5,877,016; and5,844,092, each of which is incorporated by reference in its entirety.

Further examples of Trk inhibitors include CEP-751, described in Int. J.Cancer 72:672-679, 1997; CT327, described in Acta Derm. Venereol.95:542-548, 2015; compounds described in International Publication No.WO 2012/034095; compounds described in U.S. Pat. No. 8,673,347 andInternational Publication No. WO 2007/022999; compounds described inU.S. Pat. No. 8,338,417; compounds described in InternationalPublication No. WO 2016/027754; compounds described in U.S. Pat. No.9,242,977; compounds described in U.S. Publication No. 2016/0000783;sunitinib(N-(2-diethylaminoethyl)-5-[(Z)-(5-fluoro-2-oxo-1H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide),as described in PLoS One 9:e95628, 2014; compounds described inInternational Publication No. WO 2011/133637; compounds described inU.S. Pat. No. 8,637,256; compounds described in Erpert. Opin. Ther. Pat.24(7):731-744, 2014; compounds described in Expert Opin. Ther. Pat.19(3):305-319, 2009; (R)-2-phenylpyrrolidine substitutedimidazopyridazines, e.g., GNF-8625,(R)-1-(6-(6-(2-(3-fluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-yl)-[2,4′-bipyridin]-2′-yl)piperidin-4-olas described in ACS Med. Chem. Lett. 6(5):562-567, 2015; GTx-186 andothers, as described in PLoS One 8(12):e83380, 2013; K252a ((9S-(9α,10β,12α))-2,3,9,10,11,12-hexahydro-10-hydroxy-10-(methoxycarbonyl)-9-methyl-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]benzodiazocin-1-one),as described in Mol. Cell Biochem. 339(1-2):201-213, 2010;4-aminopyrazolylpyrimidines, e.g., AZ-23(((S)-5-chloro-N2-(1-(5-fluoropyridin-2-yl)ethyl)-N4-(5-isopropoxy-1H-pyrazol-3-yl)pyrimidine-2,4-diamine)),as described in J. Med. Chem. 51(15):4672-4684, 2008; PHA-739358(danusertib), as described in Mol. Cancer Ther. 6:3158, 2007; Gö 6976(5,6,7,13-tetrahydro-13-methyl-5-oxo-12H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-12-propanenitrile),as described in J. Neurochem. 72:919-924, 1999; GW441756((3Z)-3-[(1-methylindol-3-yl)methylidene]-1H-pyrrolo[3,2-b]pyridin-2-one),as described in IJAE 115:117, 2010; milciclib (PHA-848125AC), describedin J. Carcinog. 12:22, 2013; AG-879((2E)-3-[3,5-Bis(1,1-dimethylethyl)-4-hydroxyphenyl]-2-cyano-2-propenethioamide);altiratinib(N-(4-((2-(cyclopropanecarboxamido)pyridin-4-yl)oxy)-2,5-difluorophenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide);cabozantinib(N-(4-((6,7-Dimethoxyquinolin-4-yl)oxy)phenyl)-N′-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide);lestaurtinib ((5S,6S,8R)-6-Hydroxy-6-(hydroxymethyl)-5-methyl-7,8,14,15-tetrahydro-5H-16-oxa-4b,8a,14-triaza-5,8-methanodibenzo[b,h]cycloocta[jkl]cyclopenta[e]-as-indacen-13(6H)-one);dovatinib(4-amino-5-fluoro-3-[6-(4-methylpiperazin-1-yl)-1H-benzimidazol-2-yl]quinolin-2(1H)-onemono 2-hydroxypropanoate hydrate); sitravatinib(N-(3-fluoro-4-((2-(5-(((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide);ONO-5390556; regorafenib(4-[4-({[4-Chloro-3-(trifluoromethyl)phenyl]carbamoyl}amino)-3-fluorophenoxy]-N-methylpyridine-2-carboxamidehydrate); and VSR-902A; all of the references above are incorporated byreference in their entireties herein.

The ability of a Trk inhibitor to act as a TrkA, TrkB, and/or Trk Cinhibitor may be tested using the assays described in Examples A and Bin U.S. Pat. No. 8,513,263, which is incorporated herein by reference.

In some embodiments, signal transduction pathway inhibitors includeRas-Raf-MEK-ERK pathway inhibitors (e.g., binimetinib, selumetinib,encorafinib, sorafenib, trametinib, and vemurafenib), PI3K-Akt-mTOR-S6Kpathway inhibitors (e.g. everolimus, rapamycin, perifosine,temsirolimus), and other kinase inhibitors, such as baricitinib,brigatinib, capmatinib, danusertib, ibrutinib, milciclib, quercetin,regorafenib, ruxolitinib, semaxanib, AP32788, BLU285, BLU554, INCB39110,INCB40093, INCB50465, INCB52793, INCB54828, MGCD265, NMS-088,NMS-1286937, PF 477736((R)-amino-N-[5,6-dihydro-2-(1-methyl-1H-pyrazol-4-yl)-6-oxo-1Hpyrrolo[4,3,2-ef][2,3]benzodiazepin-8-yl]-cyclohexaneacetamide),PLX3397, PLX7486, PLX8394, PLX9486, PRN1008, PRN1371, RXDX103, RXDX106,RXDX108, and TGI01209(N-tert-butyl-3-(5-methyl-2-(4-(4-methylpiperazin-1-yl)phenylamino)pyrimidin-4-ylamino)benzenesulfonamide).

Non-limiting examples of checkpoint inhibitors include ipilimumab,tremelimumab, nivolumab, pidilizumab, MPDL3208A, MEDI4736, MSB0010718C,BMS-936559, BMS-956559, BMS-935559 (MDX-1105), AMP-224, andpembrolizumab.

In some embodiments, cytotoxic chemotherapeutics are selected fromarsenic trioxide, bleomycin, cabazitaxel, capecitabine, carboplatin,cisplatin, cyclophosphamide, cytarabine, dacarbazine, daunorubicin,docetaxel, doxorubicin, etoposide, fluorouracil, gemcitabine,irinotecan, lomustine, methotrexate, mitomycin C, oxaliplatin,paclitaxel, pemetrexed, temozolomide, and vincristine.

Non-limiting examples of angiogenesis-targeted therapies includeaflibercept and bevacizumab.

In some embodiments, immune-targeted agents are selected fromaldesleukin, interferon alfa-2b, ipilimumab, lambrolizumab, nivolumab,prednisone, and sipuleucel-T.

Non-limiting examples of radiotherapy include radioiodide therapy,external-beam radiation, and radium 223 therapy.

Additional kinase inhibitors include those described in, for example,U.S. Pat. Nos. 7,514,446; 7,863,289; 8,026,247; 8,501,756; 8,552,002;8,815,901; 8,912,204; 9,260,437; 9,273,051; U.S. Publication No. US2015/0018336; International Publication No. WO 2007/002325; WO2007/002433; WO 2008/080001; WO 2008/079906; WO 2008/079903; WO2008/079909; WO 2008/080015; WO 2009/007748; WO 2009/012283; WO2009/143018; WO 2009/143024; WO 2009/014637; 2009/152083; WO2010/111527; WO 2012/109075; WO 2014/194127; WO 2015/112806; WO2007/110344; WO 2009/071480; WO 2009/118411; WO 2010/031816; WO2010/145998; WO 2011/092120; WO 2012/101032; WO 2012/139930; WO2012/143248; WO 2012/152763; WO 2013/014039; WO 2013/102059; WO2013/050448; WO 2013/050446; WO 2014/019908; WO 2014/072220; WO2014/184069; and WO 2016/075224, all of which are hereby incorporated byreference in their entireties.

Further examples of kinase inhibitors include those described in, forexample, WO 2016/081450; WO 2016/022569; WO 2016/011141; WO 2016/011144;WO 2016/011147; WO 2015/191667; WO 2012/101029; WO 2012/113774; WO2015/191666; WO 2015/161277; WO 2015/161274; WO 2015/108992; WO2015/061572; WO 2015/058129; WO 2015/057873; WO 2015/017528;WO/2015/017533; WO 2014/160521; and WO 2014/011900, each of which ishereby incorporated by reference in its entirety.

Yet other additional therapeutic agents include RET inhibitors such asthose described, for example, in U.S. Pat. Nos. 8,299,057; 8,399,442;8,937,071; 9,006,256; and 9,035,063; U.S. Publication Nos. 2014/0121239;2011/0053934; 2011/0301157; 2010/0324065; 2009/0227556; 2009/0130229;2009/0099167; 2005/0209195; International Publication Nos. WO2014/184069; WO 2014/072220; WO 2012/053606; WO 2009/017838; WO2008/031551; WO 2007/136103; WO 2007/087245; WO 2007/057399; WO2005/051366; and WO 2005/044835; and J. Med. Chem. 2012, 55 (10),4872-4876.

These additional therapeutic agents may be administered with one or morecompounds provided herein as part of the same or separate dosage forms,via the same or different routes of administration, and on the same ordifferent administration schedules according to standard pharmaceuticalpractice known to one skilled in the art.

Also provided herein is (i) a pharmaceutical combination for treatingcancer (e.g., a Trk-associated cancer) in a pediatric patient in needthereof, which comprises (a) compound of formula (I), a pharmaceuticallyacceptable salt thereof, or a combination thereof, (b) an additionaltherapeutic agent and (c) optionally at least one pharmaceuticallyacceptable carrier for simultaneous, separate or sequential use for thetreatment of a tumor disease, wherein the amounts of the compound orsalt thereof and of the additional therapeutic agent are togethereffective in treating said cancer; (ii) a pharmaceutical compositioncomprising such a combination; (iii) the use of such a combination forthe preparation of a medicament for the treatment of cancer (e.g., aTrk-associated cancer); and (iv) a commercial package or productcomprising such a combination as a combined preparation forsimultaneous, separate or sequential use; and to a method of treatmentof cancer (e.g., Trk-associated cancer) in a pediatric patient in needthereof.

Also provided are methods of treating a pediatric subject identified ordiagnosed as having a Trk-associated cancer (e.g., a subject that hasbeen identified or diagnosed as having a Trk-associated cancer throughthe use of a regulatory agency-approved, e.g., FDA-approved, kit foridentifying dysregulation of a NTRK gene, a Trk protein, or expressionor activity, or level of the same, in a pediatric subject or a biopsysample from the pediatric subject) (e.g., any of the Trk-associatedcancers described herein or known in the art) that include administeringthe pediatric subject a therapeutically effective amount of compound offormula (I), a pharmaceutically acceptable salt thereof, or acombination thereof. Also provided is a the use of a compound of formula(I), a pharmaceutically acceptable salt thereof, or a combinationthereof for use in treating a Trk-associated cancer in a pediatricsubject identified or diagnosed as having a Trk-associated cancer (e.g.,a pediatric subject that has been identified or diagnosed as having aTrk-associated cancer through the use of a regulatory agency-approved,e.g., FDA-approved, kit for identifying dysregulation of a NTRK gene, aTrk protein, or expression or activity, or level of the same, in apediatric subject or a biopsy sample from the pediatric subject) (e.g.,any of the Trk-associated cancers described herein or known in the art).Also provided is the use of compound of formula (I), a pharmaceuticallyacceptable salt thereof, or a combination thereof for the manufacture ofa medicament for treating a Trk-associated cancer in a pediatric subjectidentified or diagnosed as having a Trk-associated cancer (e.g., apediatric subject that has been identified or diagnosed as having aTrk-associated cancer through the use of a regulatory agency-approved,e.g., FDA-approved, kit for identifying dysregulation of a NTRK gene, aTrk protein, or expression or activity, or level of the same, in apediatric subject or a biopsy sample from the pediatric subject) (e.g.,any of the Trk-associated cancers described herein or known in the art).

Also provided are methods of treating a pediatric subject (e.g., apediatric subject suspected of having a Trk-associated cancer, apediatric subject presenting with one or more symptoms of aTrk-associated cancer, or a pediatric subject having an elevated risk ofdeveloping a Trk-associated cancer) that include performing an assay(e.g., an assay that utilizes next generation sequencing,immunohistochemistry, break apart FISH, or dual-fusion FISH analysis)(e.g., using a regulatory agency-approved, e.g., FDA-approved, kit) on asample obtained from the pediatric subject to determine whether thepediatric subject has dysregulation of a NTRK gene, a Trk protein, orexpression or activity, or level of the same, and administering (e.g.,specifically or selectively administering) a therapeutically effectiveamount of compound of formula (I), a pharmaceutically acceptable saltthereof, or a combination thereof to a pediatric subject determined tohave dysregulation of a NTRK gene, a Trk protein, or expression oractivity, or levels of the same. Additional assays, non-limiting assaysthat may be used in these methods are described herein. Additionalassays are also known in the art.

In some embodiments provided herein, the sample can be a tissue samplesuch as a cancer tissue sample, a biopsy sample, a serum sample, aspinal fluid sample, or a urine sample.

Also provided is use of a compound of formula (I), a pharmaceuticallyacceptable salt thereof, or a combination thereof for use in treating aTrk-associated cancer in a pediatric subject identified or diagnosed ashaving a Trk-associated cancer through a step of performing an assay(e.g., an in vitro assay) (e.g., an assay that utilizes next generationsequencing, immunohistochemistry, break apart FISH, or dual-fusion FISHanalysis) (e.g., using a regulatory agency-approved, e.g., FDA-approved,kit) on a sample obtained from the pediatric subject to determinewhether the pediatric subject has dysregulation of a NTRK gene, a Trkprotein, or expression or activity, or level of the same, where thepresence of dysregulation of a NTRK gene, a Trk protein, or expressionor activity, or level of the same, identifies that the pediatric subjecthas a Trk-associated cancer. Also provided is the use of crystallineform (I-HS) or a compound of formula (I) or a salt thereof, such as ahydrogen sulfate salt (e.g., see Example 14A of U.S. Pat. No. 8,513,263)for the manufacture of a medicament for treating a Trk-associated cancerin a pediatric subject identified or diagnosed as having aTrk-associated cancer through a step of performing an assay (e.g., an invitro assay) (e.g., an assay that utilizes next generation sequencing,immunohistochemistry, break apart FISH, or dual-fusion FISH analysis)(e.g., using a regulatory agency-approved, e.g., FDA-approved, kit) on asample obtained from the pediatric subject to determine whether thepediatric subject has dysregulation of a NTRK gene, a Trk protein, orexpression or activity, or level of the same, where the presence ofdysregulation of a NTRK gene, a Trk protein, or expression or activity,or level of the same, identifies that the pediatric subject has aTrk-associated cancer. Some embodiments of any of the methods or usesdescribed herein further include recording in the pediatric subject'sclinical record (e.g., a computer readable medium) that the pediatricsubject determined to have dysregulation of a NTRK gene, a Trk protein,or expression or activity, or level of the same, through the performanceof the assay, should be administered a compound of formula (I), apharmaceutically acceptable salt thereof, or a combination thereof.

In some embodiments of any of the methods or uses described herein, thepediatric subject has been identified or diagnosed as having a cancerwith dysregulation of a NTRK gene, a Trk protein, or expression oractivity, or level of the same (e.g., as determined using a regulatoryagency-approved, e.g., FDA-approved, assay or kit). In some embodimentsof any of the methods or uses described herein, the pediatric subjecthas a tumor that is positive for dysregulation of a NTRK gene, a Trkprotein, or expression or activity, or level of the same (e.g., asdetermined using a regulatory agency-approved assay or kit). In someembodiments of any of the methods or uses described herein, thepediatric subject can be a subject with a tumor(s) that is positive fordysregulation of a NTRK gene, a Trk protein, or expression or activity,or level of the same (e.g., identified as positive using a regulatoryagency-approved, e.g., FDA-approved, assay or kit). In some embodimentsof any of the methods or uses described herein, the pediatric subjectcan be a subject whose tumors have dysregulation of a NTRK gene, a Trkprotein, or expression or activity, or a level of the same (e.g., wherethe tumor is identified as such using a regulatory agency-approved,e.g., FDA-approved, kit or assay). In some embodiments of any of themethods or uses described herein, the pediatric subject is suspected ofhaving a Trk-associated cancer. In some embodiments of any of themethods or uses described herein, the pediatric subject has a clinicalrecord indicating that the pediatric subject has a tumor that hasdysregulation of a NTRK gene, a Trk protein, or expression or activity,or level of the same (and optionally the clinical record indicates thatthe pediatric subject should be treated with any of the compositionsprovided herein).

Also provided are methods of treating a pediatric subject that includeadministering a therapeutically effective amount of a compound offormula (I), a pharmaceutically acceptable salt thereof, or acombination thereof to a pediatric subject having a clinical record thatindicates that the pediatric subject has dysregulation of a NTRK gene, aTrk protein, or expression or activity, or level of the same. Alsoprovided is the use of a compound of formula (I), a pharmaceuticallyacceptable salt thereof, or a combination thereof for the manufacture ofa medicament for treating a Trk-associated cancer in a pediatric subjecthaving a clinical record that indicates that the pediatric subject hasdysregulation of a NTRK gene, a Trk protein, or expression or activity,or level of the same. Also provided is the use of a compound of formula(I), a pharmaceutically acceptable salt thereof, or a combinationthereof for the manufacture of a medicament for treating aTrk-associated cancer in a pediatric subject having a clinical recordthat indicates that the pediatric subject has dysregulation of a NTRKgene, a Trk protein, or expression or activity, or level of the same.Some embodiments of these methods and uses can further include: a stepof performing an assay (eg., an in vitro assay) (e.g., an assay thatutilizes next generation sequencing, immunohistochemistry, break apartFISH, or dual-fusion FISH analysis) (e.g., using a regulatoryagency-approved, e.g., FDA-approved, kit) on a sample obtained from thepediatric subject to determine whether the subject has dysregulation ofa NTRK gene, a Trk protein, or expression or activity, or level of thesame, and recording information in a pediatric subject's clinical file(e.g., a computer-readable medium) that the pediatric subject has beenidentified to have dysregulation of a NTRK gene, a Trk protein, orexpression or activity, or level of the same.

Also provided are methods (e.g., in vitro methods) of selecting atreatment for a pediatric subject that include selecting a treatmentincluding administration of a therapeutically effective amount of acompound of formula (I), a pharmaceutically acceptable salt thereof, ora combination thereof for a pediatric subject identified or diagnosed ashaving a Trk-associated cancer (e.g., a pediatric subject that has beenidentified or diagnosed as having a Trk-associated cancer through theuse of a regulatory agency-approved, e.g., FDA-approved, kit foridentifying dysregulation of a NTRK gene, a Trk protein, or expressionor activity, or level of the same, in a pediatric subject or a biopsysample from the pediatric subject) (e.g., any of the Trk-associatedcancers described herein or known in the art). Some embodiments canfurther include administering the selected treatment to the pediatricsubject identified or diagnosed as having a Trk-associated cancer. Someembodiments can further include a step of performing an assay (e.g., anin vitro assay) (e.g., an assay that utilizes next generationsequencing, immunohistochemistry, break apart FISH, or dual-fusion FISHanalysis) (e.g., using a regulatory agency-approved, e.g., FDA-approved,kit) on a sample obtained from the pediatric subject to determinewhether the pediatric subject has dysregulation of a NTRK gene, a Trkprotein, or expression or activity, or level of the same, andidentifying or diagnosing a pediatric subject determined to havedysregulation of a NTRK gene, a Trk protein, or expression or activity,or level of the same, as having a Trk-associated cancer.

Also provided are methods of selecting a treatment for a pediatricsubject that include administration of a therapeutically effectiveamount of a compound of formula (I), a pharmaceutically acceptable saltthereof, or a combination thereof, wherein the methods include a step ofperforming an assay (e.g., an in vitro assay) (e.g., an assay thatutilizes next generation sequencing, immunohistochemistry, break apartFISH, or dual-fusion FISH analysis) (e.g., using a regulatoryagency-approved, e.g., FDA-approved, kit) on a sample obtained from thepediatric subject to determine whether the subject has dysregulation ofa NTRK gene, a Trk protein, or expression or activity, or level of thesame, and identifying or diagnosing a pediatric subject determined tohave dysregulation of a NTRK gene, a Trk protein, or expression oractivity, or level of the same, as having a Trk-associated cancer, andselecting a therapeutic treatment including administration of atherapeutically effective amount of a compound of formula (I), apharmaceutically acceptable salt thereof, or a combination thereof forthe pediatric subject identified or diagnosed as having a Trk-associatedcancer. Some embodiments further include administering the selectedtreatment to the pediatric subject identified or diagnosed as having aTrk-associated cancer.

Also provided are methods of selecting a pediatric subject for treatmentincluding administration of a therapeutically effective amount of acompound of formula (I), a pharmaceutically acceptable salt thereof, ora combination thereof that include selecting, identifying, or diagnosinga pediatric subject having a Trk-associated cancer, and selecting thepediatric subject for treatment including administration of atherapeutically effective amount of a compound of formula (I), apharmaceutically acceptable salt thereof, or a combination thereof. Insome embodiments, identifying or diagnosing a pediatric subject ashaving a Trk-associated cancer can include a step of performing an assay(e.g., an in vitro assay) (e.g., an assay that utilizes next generationsequencing, immunohistochemistry, break apart FISH, or dual-fusion FISHanalysis) (e.g., using a regulatory agency-approved, e.g., FDA-approved,kit) on a sample obtained from the pediatric subject to determinewhether the subject has dysregulation of a NTRK gene, a Trk protein, orexpression or activity, or level of the same, and identifying ordiagnosing a pediatric subject determined to have dysregulation of aNTRK gene, a Trk protein, or expression or activity, or level of thesame, as having a Trk-associated cancer. In some embodiments, theselecting a treatment can be used as part of a clinical study thatincludes administration of various treatments of a Trk-associatedcancer.

In some embodiments of any of the methods or uses described herein, anassay used determine whether the pediatric subject (e.g., an infant, achild, or an adolescent) has dysregulation of a NTRK gene, a Trkprotein, or expression or activity, or level of the same, using a sample(e.g., a biological sample or a biopsy sample (e.g., a paraffin-embeddedbiopsy sample) from a subject (e.g., a pediatric subject (e.g., aninfant, a child, or an adolescent) suspected of having a Trk-associatedcancer, a pediatric subject (e.g., an infant, a child, or an adolescent)having one or more symptoms of a Trk-associated cancer, and/or apediatric subject (e.g., an infant, a child, or an adolescent) that hasan increased risk of developing a Trk-associated cancer) can include,for example, next generation sequencing, immunohistochemistry,fluorescence microscopy, break apart FISH analysis, Southern blotting,Western blotting, FACS analysis, Northern blotting, and PCR-basedamplification (e.g., RT-PCR). As is well-known in the art, the assaysare typically performed, e.g., with at least one labelled nucleic acidprobe or at least one labelled antibody or antigen-binding fragmentthereof. Assays can utilize other detection methods known in the art fordetecting dysregulation of a NTRK gene, a Trk protein, or expression oractivity, or levels of the same (see, e.g., the references citedherein).

In some embodiments, the subject (e.g., a pediatric subject, e.g., aninfant, child, or adolescent) has been identified or diagnosed as havinga cancer with dysregulation of a NTRK gene, a Trk protein, or expressionor activity, or level of the same (e.g., as determined using aregulatory agency-approved, e.g., FDA-approved, assay or kit). In someembodiments, the subject (e.g., a pediatric subject, e.g., an infant,child, or adolescent) has a tumor that is positive for dysregulation ofa NTRK gene, a Trk protein, or expression or activity, or level of thesame (e.g., as determined using a regulatory agency-approved assay orkit). The subject (e.g., a pediatric subject, e.g., an infant, child, oradolescent) can be a subject with a tumor(s) that is positive fordysregulation of a NTRK gene, a Trk protein, or expression or activity,or level of the same (e.g., identified as positive using a regulatoryagency-approved, e.g., FDA-approved, assay or kit). The subject (e.g., apediatric subject, e.g., an infant, child, or adolescent) can be asubject whose tumors have dysregulation of a NTRK gene, a Trk protein,or expression or activity, or a level of the same (e.g., where the tumoris identified as such using a regulatory agency-approved, e.g.,FDA-approved, kit or assay). In some embodiments, the subject (e.g., apediatric subject, e.g., an infant, child, or adolescent) is suspectedof having a Trk-associated cancer. In some embodiments, the subject(e.g., a pediatric subject, e.g., an infant, child, or adolescent) has aclinical record (e.g., a computer-readable medium) indicating that thesubject has a tumor that has dysregulation of a NTRK gene, a Trkprotein, or expression or activity, or level of the same (and optionallythe clinical record further indicates that the subject should be treatedwith any of the compositions provided herein).

In some embodiments, a dose contains, per unit dosage unit, about 2 mg,about 4 mg, about 6 mg, about 8 mg, about 10 mg, about 12 mg, about 14mg, about 16 mg, about 18 mg, about 20 mg, about 30 mg, about 40 mg,about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg,about 300 mg, about 400 mg, or about 500 mg of a compound of Formula(I), a pharmaceutically acceptable salt thereof, or a combinationthereof. In some embodiments, a unit dosage unit of about 2 mg to about4 mg is formulated for a one-month old patient. In some embodiments aunit dosage unit of about 6 to about 18 mg (e.g., about 6 mg, about 8mg, about 10 mg, about 12 mg, about 14 mg, about 16 mg, or about 18 mg)is formulated for a two-month or older infant. The dosages, however, maybe varied depending upon the requirement of the patients, the severityof the condition being treated and the compound being employed. In someembodiments, the dosages are administered once daily (QD) or twice daily(BID).

The daily dosage of a compound of Formula (I), a pharmaceuticallyacceptable salt thereof, or a combination thereof in a liquidformulation as described herein may be varied over a wide range from 1.0to 10,000 mg per day, or higher, or any range therein. For oraladministration, the compositions are preferably provided in the form oftablets containing, 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0,25.0, 50.0, 100, 150, 200, 250 and 500 milligrams of the activeingredient for the symptomatic adjustment of the dosage to the patientto be treated. An effective amount of the drug is ordinarily supplied ata dosage level of from about 0.1 mg/kg to about 1000 mg/kg of bodyweight per day, or any range therein. The range can be from about 0.5 toabout 500 mg/kg of body weight per day, or any range therein. The rangecan be from about 1.0 to about 250 mg/kg of body weight per day, or anyrange therein. The range can be from about 0.1 to about 100 mg/kg ofbody weight per day, or any range therein. In an example, the range maybe from about 0.1 to about 50.0 mg/kg of body weight per day, or anyamount or range therein. In another example, the range may be from about0.1 to about 15.0 mg/kg of body weight per day, or any range therein. Inyet another example, the range may be from about 0.5 to about 7.5 mg/kgof body weight per day, or any amount to range therein. A liquidformulation as provided herein may be administered on a regimen of 1 to4 times per day or in a single daily dose

Optimal dosages to be administered may be readily determined by thoseskilled in the art, and will vary with the mode of administration, thestrength of the preparation, the mode of administration, and theadvancement of the disease condition. In addition, factors associatedwith the particular patient being treated, including patient age,weight, diet and time of administration, will result in the need toadjust dosages.

In some embodiments, the compounds and formulations provided herein areadministered on a continuous 28-day schedule. For example, a singlecycle of administration includes 28 days of continuous dosing. Suchdosing can be, for example, one daily or twice daily.

One skilled in the art will recognize that, both in vivo and in vitrotrials using suitable, known and generally accepted cell and/or animalmodels are predictive of the ability of a test compound to treat orprevent a given disorder.

One skilled in the art will further recognize that human clinical trialsincluding first-in-human, dose ranging and efficacy trials, in healthypatients and/or those suffering from a given disorder, may be completedaccording to methods well known in the clinical and medical arts.

In some embodiments, the methods provided can follow after surgicalresection has failed to inhibit progression of the fibrosarcoma in thesubject. The methods provided herein can also follow after chemotherapyincluding administration of at least one of vincristine, actinomycin-D,cyclophosphamide, ifosfamide, etoposide, doxorubicin has failed toinhibit tumor progression in the subject. For example, the methodsprovided herein can follow after administration of at least one ofvincristine, actinomycin-D, and cyclophosphamide has failed to inhibittumor progression in the subject. The methods provided herein can alsofollow after administration of at least one of ifosfamide anddoxorubicin has failed to inhibit tumor progression in the subject.

Methods of Treating a Pediatric Cancer with a Crystalline Form of aCompound of Formula (I)

For the methods of treatment provided herein, a compound of formula (I),or a pharmaceutically acceptable salt thereof, can be provided in acrystalline form.

For example, a crystalline form of the compound of formula (I) caninclude the hydrogen sulfate salt of the compound of formula (I) in astable polymorph form, hereinafter referred to as crystalline form(I-HS), which may be characterized, for example, by its X-raydiffraction pattern.

As illustrated in FIG. 1, in some embodiments, the crystalline form(I-HS) can be characterized by its X-ray powder diffraction pattern(XRPD). The XRPD was carried out on a D5000 X-ray diffractometer with aCuKα1, 0.1540562 nm long, fine focus sealed tube source from Siemens byscanning samples between 3 and 40°2-theta at a step size of0.0200°2-theta and a time per step of 1 second. The effective scan speedwas 0.0200°/s with an instrument voltage 40 kV and a current setting of40 mA. Samples were analyzed using a divergence slit having a size of 2mm in reflection mode under the following experimental conditions.

In some embodiments, crystalline form (I-HS) has an XRPD pattern with atleast the characteristic peaks (2θ degrees±0.3), as listed in Table 1.

TABLE 1 XRPD peaks of crystalline form (I-HS) Relative Position [°2-θ]FWHM [°2-θ] d-spacing [Å] Intensity [%] 10.63 0.12 8.32 27.44 15.25 0.145.81 12.24 16.39 0.13 5.40 13.92 18.37 0.13 4.82 43.65 19.08 0.14 4.6519.60 19.79 0.11 4.48 9.83 20.15 0.25 4.40 25.09 20.61 0.13 4.31 100.0021.47 0.21 4.14 24.71 22.01 0.12 4.03 14.45 23.04 0.15 3.86 33.01 23.970.12 3.71 38.52 24.35 0.21 3.65 10.05 25.58 0.13 3.48 8.11 26.48 0.173.36 9.76 27.50 0.14 3.24 7.70 28.17 0.17 3.16 11.60 28.58 0.19 3.1210.85 30.77 0.29 2.90 8.48 38.47 0.21 2.34 10.97

In some embodiments, the crystalline form (I-HS) has an XRPD patternwith at least the 8 characteristic peaks (2θ degrees±0.3), whichcomprises peaks having a relative intensity greater than or equal toabout 15%, as listed in Table 2.

TABLE 2 XRPD peaks of crystalline form (I-HS) Relative Position [°2-θ]FWHM [°2-θ] d-spacing [Å] Intensity [%] 10.63 0.12 8.32 27.44 18.37 0.134.82 43.65 19.08 0.14 4.65 19.60 20.15 0.25 4.40 25.09 20.61 0.13 4.31100.00 21.47 0.21 4.14 24.71 23.04 0.15 3.86 33.01 23.97 0.12 3.71 38.52

In some embodiments, the crystalline form (I-HS) has an XRPD patternwith at least the 5 characteristic peaks (2θ degrees±0.3), whichcomprises peaks having a relative intensity greater than or equal toabout 25%, as listed in Table 3.

TABLE 3 XRPD peaks of crystalline form (I-HS) Relative Position [°2-θ]FWHM [°2-θ] d-spacing [Å] Intensity [%] 10.63 0.12 8.32 27.44 18.37 0.134.82 43.65 20.61 0.13 4.31 100.00 23.04 0.15 3.86 33.01 23.97 0.12 3.7138.52

In some embodiments, the crystalline form (I-HS) has an XRPD patternwith at least the 4 characteristic peaks (2θ degrees±0.3), whichcomprises peaks having a relative intensity greater than or equal toabout 30%, as listed in Table 4.

TABLE 4 XRPD peaks of crystalline form (I-HS) Relative Position [°2-θ]FWHM [°2-θ] d-spacing [Å] Intensity [%] 18.37 0.13 4.82 43.65 20.61 0.134.31 100.00 23.04 0.15 3.86 33.01 23.97 0.12 3.71 38.52

In certain embodiments, crystalline form (I-HS) has an XRPD pattern thatis substantially the same XRPD pattern as shown in FIG. 1.

In some embodiments, crystalline form (I-HS) is characterized by havingXRPD diffraction peaks (2θ degrees) at about 18.4, 20.6, 23.0, and 24.0.In some embodiments, crystalline form (I-HS) is characterized by havingXRPD diffraction peaks (2θ degrees) at about 10.6, 18.4, 20.6, 23.0, and24.0. In some embodiments, crystalline form (I-HS) is characterized byhaving XRPD diffraction peaks (2θ degrees) at about 10.6, 18.4, 19.1,20.2, 20.6, 21.5, 23.0, and 24.0. In some embodiments, crystalline form(I-HS) is characterized by having XRPD diffraction peaks (2θ degrees) atabout 10.6, 15.3, 16.4, 18.4, 19.1, 19.8, 20.2, 20.6, 21.5, 22.0, 23.0,24.0, 24.4, 25.6, 26.5, 27.5, 28.2, 28.6, 30.8, and 38.5.

In certain embodiments, crystalline form (I-HS) has an XRPD pattern thatis substantially the same XRPD pattern as shown in FIG. 8.

In some embodiments, crystalline form (I-HS) has an XRPD pattern with atleast the 20 characteristic peaks (2θ degrees±0.3), as listed in Table1.

TABLE 5 XRPD peaks of crystalline form (I-HS) Position (°2θ) RelativeIntensity (%) 10.76 29.85 15.38 13.22 16.52 16.46 18.50 48.07 19.2222.92 19.92 16.05 20.26 30.80 20.74 100.00 21.56 23.78 22.16 15.51 23.1632.52 24.10 33.89 24.50 12.14 25.72 8.89 26.50 10.88 27.62 8.61 28.3211.44 28.74 10.73 30.92 8.23 38.60 8.88

In some embodiments, the crystalline form (I-HS) has an XRPD patternwith at least the 8 characteristic peaks (2θ degrees±0.3), whichcomprises peaks having a relative intensity greater than or equal toabout 15%, as listed in Table 6.

TABLE 6 XRPD peaks of crystalline form (I-HS) Position (°2θ) RelativeIntensity (%) 10.76 29.85 18.50 48.07 19.22 22.92 20.26 30.80 20.74100.00 21.56 23.78 23.16 32.52 24.10 33.89

In some embodiments, the crystalline form (I-HS) has an XRPD patternwith at least the 5 characteristic peaks (2θ degrees±0.3), whichcomprises peaks having a relative intensity greater than or equal toabout 25%, as listed in Table 7.

TABLE 7 XRPD peaks of crystalline form (I-HS) Position (°2θ) RelativeIntensity (%) 10.76 29.85 18.50 48.07 20.74 100.00 23.16 32.52 24.1033.89

In some embodiments, the crystalline form (I-HS) has an XRPD patternwith at least the 4 characteristic peaks (2θ degrees±0.3), whichcomprises peaks having a relative intensity greater than or equal toabout 30%, as listed in Table 8.

TABLE 8 XRPD peaks of crystalline form (I-HS) Position (°2θ) RelativeIntensity (%) 18.50 48.07 20.74 100.00 23.16 32.52 24.10 33.89

In some embodiments, crystalline form (I-HS) is characterized by havingXRPD diffraction peaks (2θ degrees) at about 18.5, 20.7, 23.2, and 24.1.In some embodiments, crystalline form (I-HS) is characterized by havingXRPD diffraction peaks (2θ degrees) at about 10.8, 18.5, 20.7, 23.2, and24.1. In some embodiments, crystalline form (I-HS) is characterized byhaving XRPD diffraction peaks (2θ degrees) at about 10.8, 18.5, 19.2,20.3, 20.7, 21.6, 23.2, and 24.1. In some embodiments, crystalline form(I-HS) is characterized by having XRPD diffraction peaks (2θ degrees) atabout 10.8, 15.4, 16.5, 18.5, 19.2, 19.9, 20.3, 20.7, 21.6, 22.2, 23.2,24.1, 24.5, 25.7, 26.5, 27.6, 28.3, 28.7, 30.9, and 38.6.

In some embodiments, given the XRPD patterns provided in FIGS. 1 and 29,crystalline form (I-HS) is characterized by having XRPD peaks (2θdegrees) as shown in Table 9.

TABLE 9 XRPD peaks of crystalline form (I-HS) FIG. 1 FIG. 29 DifferenceAverage 10.76 10.63 0.13 10.70 15.38 15.25 0.13 15.32 16.52 16.39 0.1316.46 18.50 18.37 0.13 18.44 19.22 19.08 0.14 19.15 19.92 19.79 0.1319.86 20.26 20.15 0.11 20.21 20.74 20.61 0.13 20.68 21.56 21.47 0.0921.52 22.16 22.01 0.15 22.09 23.16 23.04 0.12 23.10 24.10 23.97 0.1324.04 24.50 24.35 0.15 24.43 25.72 25.58 0.14 25.65 26.50 26.48 0.0226.49 27.62 27.50 0.12 27.56 28.32 28.17 0.15 28.25 28.74 28.58 0.1628.66 30.92 30.77 0.15 30.85 38.60 38.47 0.13 38.54

In some embodiments, crystalline form (I-HS) is characterized by havingXRPD diffraction peaks (2θ degrees) at 18.4±0.2, 20.7±0.2, 23.1±0.2, and24.0±0.2. In some embodiments, crystalline form (I-HS) is characterizedby having XRPD diffraction peaks (20 degrees) at 10.7±0.2, 18.4±0.2,20.7±0.2, 23.1±0.2, and 24.0±0.2. In some embodiments, crystalline form(I-HS) is characterized by having XRPD diffraction peaks (2θ degrees) at10.7±0.2, 18.4±0.2, 19.2±0.2, 20.2±0.2, 20.7±0.2, 21.5±0.2, 23.1±0.2,and 24.0±0.2. In some embodiments, crystalline form (I-HS) ischaracterized by having XRPD diffraction peaks (20 degrees) at 10.7±0.2,15.3±0.2, 16.5±0.2, 18.4±0.2, 19.2±0.2, 19.9±0.2, 20.2±0.2, 20.7±0.2,21.5±0.2, 22.1±0.2, 23.1±0.2, 24.0±0.2, 24.4±0.2, 25.6±0.2, 26.5±0.2,27.6±0.2, 28.2±0.2, 28.7±0.2, 30.8±0.2, and 38.5±0.2.

It will be understood that the 2-theta values of the X-ray powderdiffraction patterns for crystalline form (I-HS) may vary slightly fromone instrument to another and also depending on variations in samplepreparation and batch to batch variation, and so the values quoted arenot to be construed as absolute. It will also be understood that therelative intensities of peaks may vary depending on orientation effectsso that the intensities shown in the XRPD trace included herein areillustrative and not intended to be used for absolute comparison.Accordingly, it is to be understood that the phrase “substantially thesame XRPD pattern as shown in FIG. 1 or FIG. 8” means that forcomparison purposes, at least 90% of the peaks shown in FIG. 1 or FIG. 8are present. It is to be understood that the relative peak positions mayvary ±0.3 degrees from the peak positions shown in FIG. 1 or FIG. 8. Itis to be further understood that for comparison purposes somevariability in peak intensities from those shown in FIG. 1 and FIG. 8 isallowed.

FIG. 2 illustrates a simultaneous thermogravimetric/differential thermalanalyzer (TG/DTA) profile of crystalline form (I-HS), according to oneembodiment. For the analysis about 5 mg of crystalline form (I-HS) wasweighed into an open aluminum pan and loaded into a simultaneousthermogravimetric/differential thermal analyzer (TG/DTA) and held atroom temperature. The sample was then heated at a rate of 10°Celsius/min from 25° Celsius to 300° Celsius during which time thechange in sample weight was recorded along with any differential thermalevents. Nitrogen was used as the purge gas at a flow rate of 100cm³/min. The TG/DAT profile of crystalline form (I-HS) shows an initialweight loss of 0.8% between 27.4° Celsius to 182.40 Celsius, which isfollowed by 4.9% weight loss in the TG curve between 182.4° Celsius to225.00 Celsius, also seen as an endotherm in the DTA curve. These weightlosses could be decomposition of the material.

FIG. 3 illustrates a differential scanning calorimetry (DSC) profile ofcrystalline form (I-HS), according to one embodiment. DSC analysis ofthe sample was performed using a Seiko DSC6200 differential scanningcalorimeter (equipped with a cooler). About 5 mg of crystalline form(I-HS) was weighed into an aluminum DSC pan and sealed non-hermeticallywith a pierced aluminum lid. The sample pan was then loaded into a SeikoDSC6200 (equipped with a cooler), cooled, and held at 25° Celsius. Oncea stable heat-flow response was obtained, the sample and reference wereheated to 270° Celsius at a scan rate of 10° Celsius/min whilemonitoring the resulting heat flow response. In some embodiments,crystalline form (I-HS) has a DSC thermogram substantially as shown inFIG. 3. As used herein, “substantially as shown in FIG. 3” means thatthe temperatures of the endothermic event shown in FIG. 3 can vary byabout +5° C.

As shown in FIG. 3, the DSC thermogram of the crystalline form (I-HS)indicates a small endothermic change in the baseline between 122.9°Celsius to 152.80 Celsius, followed by a sharp endotherm thatcorresponds to the melting of the crystalline form (I-HS) at an onsettemperature of melting of 190.8° Celsius, a peak temperature of meltingof 197.9° Celsius and a heat of melting of 2.415 mW. The transitionfollowing the melting endotherm may be caused by the decomposition ofthe melted crystalline form (I-HS).

FIGS. 4A and 4B illustrate polarized light microscopy (PLM) images ofcrystalline form (I-HS) under (A) unpolarized and (B) unpolarized light,according to some embodiments. The presence of crystallinity(birefringence) was determined using an Olympus BX50 polarizingmicroscope, equipped with a Motic camera and image capture software(Motic Images Plus 2.0). All images were recorded using the20×objective. The crystalline form (I-HS) exhibits birefringence whenexamined under polarized light without exhibiting a definite morphologyor agglomerates.

FIG. 5 illustrates a dynamic vapor sorption (DVS) isotherm profile ofcrystalline form (I-HS), according to one embodiment. For the DVSmeasurement a sample of crystalline form (I-HS) was cycled throughchanging humidity conditions to determine its hygroscopicity. The samplewas analyzed using a Surface Measurement System DVS-1 Dynamic VaporSorption System. About 10 mg of crystalline form (I-HS) was placed intoa mesh vapor sorption balance pan and loaded into a dynamic vaporsorption balance as part of the Surface Measurement System. Data wascollected in 1 minute intervals. Nitrogen was used as the carrier gas.The sampled crystalline form (I-HS) was subjected to a ramping profilefrom 20-90% relative humidity (RH) at 10% increments, maintaining thesample at each step until a stable weight had been achieved (99.5% stepcompletion). After completion of the sorption cycle, the sample wasdried using the same procedure, but all the way down to 0% RH andfinally taken back to the starting point of 20% RH. The weight changeduring the sorption/desorption cycles were plotted, allowing for thehygroscopic nature of the sample to be determined.

As shown in FIG. 5, crystalline form (I-HS) appears to benon-hygroscopic. A small increase in mass of about 1.7% was observedbetween 0%/o and 90% RH during the sorption cycle. In addition, a verysmall hysteresis was observed between sorption and desorption cycles.The XRPD pattern of crystalline form (I-HS) post DVS analysis (notshown) being similar to its pre-DVS XRPD pattern shown in FIG. 1 or FIG.29 indicates that no change in the crystalline form (I-HS) occurredduring DVS.

FIG. 6 illustrates an infrared (IR) spectroscopy profile of crystallineform (I-HS) for the compound of formula (I), according to oneembodiment. IR spectroscopy was carried out on a Bruker ALPHA Pspectrometer. Sufficient material of crystalline form (I-HS) was placedonto the center of the plate of the spectrometer with a transmittancespectrum being obtained using a resolution of 4 cm⁻¹, a background scantime of 16 scans, a sample scan time of 16 scans, and collecting datafrom 4000 cm⁻¹ to 400 cm⁻¹. The observed IR spectrum of crystalline form(I-HS) is shown in FIG. 6.

The crystalline form (I-HS) has a number of properties that make itsurprisingly superior to the amorphous form of(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehydrogen sulfate (AM(HS)). For example, the crystalline form (I-HS) hasproperties which contribute to its manufacturability and production of acommercial product. As shown in Example 8, the crystalline form (I-HS)has better flow properties as compared to the amorphous API (AM(HS)) asevidenced by the Carr's and Hausner Index. For example, the crystallineform (I-HS) exhibits a Carr Index value of greater than 20%. In someembodiments, the crystalline form (I-HS) exhibits a Hausner ratio ofless than 1.35 (e.g., a value of between about 1.26 to about 1.34). Thedifferences in flow properties can make the development of a solid oraldosage form more difficult for the amorphous API vs. the crystallineAPI.

The crystalline form (I-HS) also evidenced better stability in anaccelerated stability study conducted in an LDPE bag at 40° C./75% RHfor five weeks. While neither the AM(HS) or crystalline form (I-HS)exhibited a significant changes in chemical impurity levels over thecourse of the study, the study did reveal that the crystalline form(I-HS) has stable physicochemical properties. The amorphous API, on theother hand, converted into a crystalline form substantially similar tothe crystalline form (I-HS) by XRPD, DSC, TGA, KF and polarized lightmicroscopy. Additionally, the amorphous API changed to an agglomeratedpowder with reduced flow properties over the course of the stabilitytesting. Such changes in the physical properties of the compound,including a change from an amorphous power to a crystalline materialand/or an agglomerated powder with reduced flow, on storage would makeit nearly impossible to manufacture a solid oral dosage form for patientuse based on the amorphous compound. The properties observed for thecrystalline form (I-HS), however, are consistent with that desired for acommercial product, including having both a stable physical and chemicalstructure.

The crystalline form (I-HS), as noted previously, is non-hygroscopic. Asused herein, “non-hygroscopic” refers to a compound exhibiting less thana 2% weight gain at 25° C. and 80% RH after 24 to 48 hours (see, e.g.,Example 10). The AM(HS) compound, however, was found to deliquesce uponexposure to humidity. Given this tendency, use of the AM(HS) compoundwould require significant handling precautions during storage andmanufacture to prevent this change in form from occurring whereas thecrystalline form (I-HS) requires no such precautions during manufactureof the API. This stability to humidity would also be expected to carryover to any solid oral dosage product prepared using the crystallineform (I-HS).

Finally, the crystalline form (I-HS) provides a significantly improvedimpurity profile versus the amorphous API. The ability to control animpurity profile is important for patient safety, developing arepeatable manufacturing process, and meeting requirements by Regulatoryagencies prior to use in humans.

The compounds provided herein, including(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide(formula (I)) and pharmaceutically acceptable salts thereof, for examplethe hydrogen sulfate salt, and further a novel crystalline form of thehydrogen sulfate salt (crystalline form (I-HS)), exhibit Trk familyprotein tyrosine kinase inhibition, and the compound, hydrogen sulfatesalt, and crystalline form thereof can be used in the treatment of pain,inflammation, cancer, and certain infectious diseases.

Non-limiting examples of doses of crystalline form (I-HS) or a compoundof formula (I) or a salt thereof, such as a hydrogen sulfate salt (e.g.,see Example 14A of U.S. Pat. No. 8,513,263) that can be administered toa pediatric subject (in any of the methods or uses described herein) aredescribed herein. Non-limiting examples of the frequency ofadministration of crystalline form (l-HS) or a compound of formula (I)or a salt thereof, such as a hydrogen sulfate salt (e.g., see Example14A of U.S. Pat. No. 8,513,263) to a pediatric subject (that can be usedin any of the methods or uses described herein) are described herein.

Also provided herein is a method of treating diseases or medicalconditions in a pediatric subject in need thereof, wherein said diseaseor condition is treatable with an inhibitor of TrkA and/or TrkB (e.g., aTrk-associated cancer), comprising administering to said subjectcrystalline form (I-HS) or a compound of formula (I) or a apharmaceutically acceptable salt thereof, such as a hydrogen sulfatesalt (e.g., see Example 14A of U.S. Pat. No. 8,513,263 in an amounteffective to treat or prevent said disorder.

Crystalline form (I-HS) or a compound of formula (I) or apharmaceutically acceptable salt thereof, such as a hydrogen sulfatesalt (e.g., see Example 14A of U.S. Pat. No. 8,513,263) can be used incombination with one or more additional drugs that work by the same or adifferent mechanism of action. Such conjoint treatment may be achievedby way of the simultaneous, sequential or separate administration of theindividual components of the treatment. Examples includeanti-inflammatory compounds, steroids (e.g., dexamethasone, cortisoneand fluticasone), analgesics such as NSAIDs (e.g., aspirin, ibuprofen,indomethacin, and ketoprofen), and opioids (such as morphine), andchemotherapeutic agents.

In the field of medical oncology, it is normal practice to use acombination of different forms of treatment to treat each pediatricpatient with cancer. In medical oncology the other component(s) of suchconjoint treatment in addition to compositions provided herein may be,for example, surgery, radiotherapy, chemotherapy, signal transductioninhibitors and/or monoclonoal antibodies.

Accordingly, crystalline form (I-HS) may be administered in combinationwith one or more agents selected from mitotic inhibitors, alkylatingagents, anti-metabolites, antisense DNA or RNA, intercalatingantibiotics, growth factor inhibitors, signal transduction inhibitors,cell cycle inhibitors, enzyme inhibitors, retinoid receptor modulators,proteasome inhibitors, topoisomerase inhibitors, biological responsemodifiers, anti-hormones, angiogenesis inhibitors, cytostatic agentsanti-androgens, targeted antibodies, HMG-CoA reductase inhibitors, andprenyl-protein transferase inhibitors.

It will be appreciated that crystalline form (I-HS) contains two centersof asymmetry and may therefore be prepared and isolated in a mixture ofisomers such as a racemic or diastereomeric mixture, or in anenantiomerically pure form. Where stereochemistry is specified by asolid wedge or dashed line representing a particular configuration, thenthat stereoisomer is so specified and defined.

Crystalline form (I-HS) may be administered by any convenient route,e.g. into the gastrointestinal tract (e.g., rectally or orally), thenose, lungs, musculature or vasculature, or transdermally or dermally.Crystalline form (I-HS) may be administered in any convenientadministrative form, e.g., tablets, powders, capsules, solutions,dispersions, suspensions, syrups, sprays, suppositories, gels,emulsions, patches etc. Such compositions may contain componentsconventional in pharmaceutical preparations, e.g. diluents, carriers, pHmodifiers, sweeteners, bulking agents, and further active agents. Ifparenteral administration is desired, the compositions will be sterileand in a solution or suspension form suitable for injection or infusion.

Methods of Treating a Pediatric Cancer with a Composition of a Compoundof Formula (I)

For the methods of treatment provided herein, a compound of formula (I),or a pharmaceutically acceptable salt thereof, can be provided as apharmaceutical composition.

Pharmaceutical compositions can include a compound of formula (I), or apharmaceutically acceptable salt thereof, or a combination thereof, anda pharmaceutically acceptable carrier. Pharmaceutical compositionscontaining a compound of formula (I), or a pharmaceutically acceptablesalt thereof, or a combination thereof, as the active ingredient can beprepared by intimately mixing a compound of formula (I), or apharmaceutically acceptable salt thereof, or a combination thereof, witha pharmaceutical carrier according to conventional pharmaceuticalcompounding techniques. The carrier may take a wide variety of formsdepending upon the desired route of administration (e.g., oral,parenteral). Solid oral preparations may be coated with substances suchas sugars or be enteric-coated so as to modulate major site ofabsorption. For parenteral administration, the carrier will usuallyconsist of sterile water and other ingredients may be added to increasesolubility of the compound or to preserve and increase the stability ofthe solution. Injectable suspensions or solutions may also be preparedutilizing aqueous carriers along with appropriate additives. The liquidformulations provided herein can be administered through a number ofdifferent routes including oral administration, intranasaladministration, and administration through an enteral feeding orgastrostomy tube (e.g., intra-jejunal tube)

Also provided herein are pharmaceutical compositions comprising acrystalline form (I-HS). To prepare the pharmaceutical compositionsprovided herein, crystalline form (I-HS) as the active ingredient isintimately admixed with a pharmaceutical carrier according toconventional pharmaceutical compounding techniques, which carrier maytake a wide variety of forms depending of the form of preparationdesired for administration, e.g., oral or parenteral such asintramuscular. In preparing the compositions in oral dosage form, any ofthe usual pharmaceutical media may be employed. For solid oralpreparations such as, for example, powders, capsules, caplets, gelcapsand tablets, suitable carriers and additives include starches, sugars,diluents, granulating agents, lubricants, binders, disintegrating agentsand the like. Suitable binders include, without limitation, starch,gelatin, natural sugars such as glucose or beta-lactose, cornsweeteners, natural and synthetic gums such as acacia, tragacanth orsodium oleate, sodium stearate, magnesium stearate, sodium benzoate,sodium acetate, sodium chloride and the like. Disintegrators include,without limitation, starch, methyl cellulose, agar, bentonite, xanthangum and the like.

In some embodiments, tablets and capsules may be sugar coated or entericcoated by standard techniques. For parenterals, the carrier can comprisesterile water, through other ingredients, for example, for purposes suchas aiding solubility or for preservation, can be included. Injectablesuspensions may also be prepared, in which case appropriate liquidcarriers, suspending agents and the like may be employed. Thepharmaceutical compositions herein can contain, per dosage unit, e.g.,tablet, capsule, powder, injection, teaspoonful and the like, an amountof the active ingredient necessary to deliver an effective dose asdescribed above.

The pharmaceutical compositions herein can contain, per unit dosageunit, e.g., tablet, capsule, suspension, solution, sachet forreconstitution, powder, injection, I.V., suppository, sublingual/buccalfilm, teaspoonful and the like, of from about 0.1-1000 mg or any rangetherein, and may be given at a dosage of from about 0.01-300 mg/kg/day,or any range therein, preferably from about 0.5-50 mg/kg/day, or anyrange therein. In some embodiments, the pharmaceutical compositionsprovided herein contain, per unit dosage unit, about 25 mg to about 500mg of a compound provided herein (for example, about 25 mg to about 400mg, about 25 mg to about 300 mg, about 25 mg to about 250 mg, about 25mg to about 200 mg, about 25 mg to about 150 mg, about 25 mg to about100 mg, about 25 mg to about 75 mg, about 50 mg to about 500 mg, about100 mg to about 500 mg, about 150 mg to about 500 mg, about 200 mg toabout 500 mg, about 250 mg to about 500 mg, about 300 mg to about 500mg, about 400 mg to about 500 mg, about 50 to about 200 mg, about 100 toabout 250 mg, about 50 to about 150 mg). In some embodiments, thepharmaceutical compositions provided herein contain, per unit dosageunit, about 25 mg, about 50 mg, about 100 mg, about 150 mg, about 200mg, about 250 mg, about 300 mg, about 400 mg, or about 500 mg of acompound provided herein. The dosages, however, may be varied dependingupon the requirement of the patients, the severity of the conditionbeing treated and the compound being employed. In some embodiments, thedosages are administered once daily (QD) or twice daily (BID).

In some embodiments, these compositions are in unit dosage forms such astablets, pills, capsules, powders, granules, sterile parenteralsolutions or suspensions, metered aerosol or liquid sprays, drops,ampoules, autoinjector devices or suppositories; for oral parenteral,intranasal, sublingual or rectal administration, or for administrationby inhalation or insufflation. Alternatively, the composition may bepresented in a form suitable for once-weekly or once-monthlyadministration; for example, an insoluble salt of the active compound,such as the decanoate salt, may be adapted to provide a depotpreparation for intramuscular injection. For preparing solidcompositions such as tablets, a compound of formula (I), apharmaceutically acceptable salt thereof, crystalline form (I-HS), or acombination thereof, is mixed with a pharmaceutical carrier, e.g.conventional tableting ingredients such as corn starch, lactose,sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalciumphosphate or gums, and other pharmaceutical diluents, e.g. water, toform a solid preformulation composition containing a homogeneous mixtureof the compound of formula (I), a pharmaceutically acceptable saltthereof, crystalline form (I-HS), or a combination thereof. Whenreferring to these preformulation compositions as homogeneous, it ismeant that the active ingredient is dispersed evenly throughout thecomposition so that the composition may be readily subdivided intoequally effective dosage forms such as tablets, pills and capsules. Thissolid preformulation composition is then subdivided into unit dosageforms of the type described above containing from 0.1 to about 1000 mg,or any amount or range thereof, of the active ingredient providedherein. The tablets or pills of the novel composition can be coated orotherwise compounded to provide a dosage form affording the advantage ofprolonged action. For example, the tablet or pill can comprise an innerdosage and an outer dosage component, the latter being in the form of anenvelope over the former. The two components can be separated by anenteric layer which serves to resist disintegration in the stomach andpermits the inner component to pass intact into the duodenum or to bedelayed in release. A variety of material can be used for such entericlayers or coatings, such materials including a number of polymeric acidswith such materials as shellac, cetyl alcohol and cellulose acetate.

A compound of formula (I), a pharmaceutically acceptable salt thereof,crystalline form (I-HS), or a combination thereof, can be administeredin intranasal form via topical use of suitable intranasal vehicles, orvia transdermal skin patches well known to those of ordinary skill inthat art. To be administered in the form of a transdermal deliverysystem, the dosage administration will, of course, be continuous ratherthan intermittent throughout the dosage regimen.

To prepare a pharmaceutical compositions provided herein, a compound offormula (I), a pharmaceutically acceptable salt thereof, crystallineform (I-HS), or a combination thereof as the active ingredient isintimately admixed with a pharmaceutical carrier according toconventional pharmaceutical compounding techniques, which carrier maytake a wide variety of forms depending of the form of preparationdesired for administration (e.g. oral or parenteral). Suitablepharmaceutically acceptable carriers are well known in the art.Descriptions of some of these pharmaceutically acceptable carriers maybe found in The Handbook of Pharmaceutical Excipients, published by theAmerican Pharmaceutical Association and the Pharmaceutical Society ofGreat Britain.

Methods of formulating pharmaceutical compositions have been describedin numerous publications such as Pharmaceutical Dosage Forms: Tablets,Second Edition, Revised and Expanded, Volumes 1-3, edited by Liebermanet al; Pharmaceutical Dosage Forms: Parenteral Medications, Volumes 1-2,edited by Avis et al; and Pharmaceutical Dosage Forms: Disperse Systems,Volumes 1-2, edited by Lieberman et al; published by Marcel Dekker, Inc.

Methods of Treating a Pediatric Cancer with a Liquid Formulation of aCompound of Formula (I)

For the methods of treatment provided herein, a compound of formula (I),a pharmaceutically acceptable salt thereof, or combinations thereof canbe provided as a liquid formulation.

Provided herein is a liquid formulation including a solubilizing agentand(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehaving the formula (I):

a pharmaceutically acceptable salt thereof, or a combination thereof.

In some embodiments, the compound of formula (I), a pharmaceuticallyacceptable salt thereof, or a combination thereof, can be present in theliquid formulation in an amount from about 0.5 wt. % to about 7 wt. %,about 1 wt. % to about 3 wt. %, or about 1.5 wt. % to about 2.5 wt. %.For example, the compound of formula (I), a pharmaceutically acceptablesalt thereof, or a combination thereof can be present in the liquidformulation in an amount of about 0.5 wt. %, 1 wt. %, 2 wt. %, 3 wt. %,4 wt. %, 5 wt. %, 6 wt. %, or about 7 wt. %. In some embodiments, thecompound of formula (I), a pharmaceutically acceptable salt thereof, ora combination thereof can be present in the liquid formulation in anamount of about 2 wt. %.

In some embodiments, the compound of formula (I), a pharmaceuticallyacceptable salt thereof, or a combination thereof, has a concentrationof about 5 mg/mL to about 50 mg/mL, about 15 mg/mL to about 35 mg/mL, orabout 20 mg/mL to about 30 mg/mL in the liquid formulation. For example,the compound of formula (I), a pharmaceutically acceptable salt thereof,or a combination thereof can have a concentration of about 5 mg/mL, 10mg/mL, 15 mg/mL, 20 mg/mL, 25 mg/mL, 30 mg/mL, 35 mg/m, 40 mg/mL, 45mg/mL, or about 50 mg/mL in the liquid formulation. In some embodiments,the compound of formula (I), a pharmaceutically acceptable salt thereof,or a combination thereof can be present at a concentration of about 20mg/mL in the liquid formulation.

The formulations provided herein can include a solubilizing agent thatfunctions to increase the solubility of the compound of formula (I), apharmaceutically acceptable salt thereof, or a combination thereof. Asolubilizing agent as provided herein is a polar organic compound havingone or more hydroxyl groups. The solubilizing agent is also capable ofachieving a higher concentration of the compound of formula (I) (e.g.,the free base) in aqueous solution compared to an aqueous phasedissolution of the compound of formula (I) in a similar pH range withoutthe solubilizing agent. The solubilizing agent can include, for example,a cyclodextrin, a glycol, a glycerol, a polyethylene glycol, aself-emulsifying drug delivery system (SEDDS), or a combination thereof.

In some embodiments, the cyclodextrin can include an α-cyclodextrin,β-cyclodextrin derivative, a δ-cyclodextrin derivative, aγ-cyclodextrin, or a combination derivative thereof.

For example, the solubilizing agent can include a cyclodextrin. Thesolubilizing agent can include a β-cyclodextrin derivative, aγ-cyclodextrin, or a mixture thereof. For example, the solubilizingagent can include a hydroxy alkyl-γ-cyclodextrin. In some embodiments,the solubilizing agent includes a β-cyclodextrin including at least oneof a hydroxy alkyl-β-cyclodextrin (e.g., hydroxypropyl-β-cyclodextrin)or a sulfoalkyl ether-J-cyclodextrin (e.g., sulfobutylether-β-cyclodextrin). For example, the liquid the solubilizing agentcan include hydroxypropyl-β-cyclodextrin. In some embodiments, thecyclodextrin is CAVASOL® W7 HP (hydroxypropyl-β-cyclodextrin). In someembodiments, the cyclodextrin is KLEPTOSE® HP(hydroxypropyl-β-cyclodextrin). In some embodiments, the cyclodextrin isCAVAMAX® W7 (β-cyclodextrin). In some embodiments, the cyclodextrin isCAPTISOL® (sulfoalkyl ether-β-cyclodextrin). In some embodiments, thecyclodextrin is CAVASOL® W7 M (methyl-β-cyclodextrin). In someembodiments, the cyclodextrin is CAVASOL® W8 HP(hydroxypropyl-γ-cyclodextrin). In some embodiments, the cyclodextrin isCAVAMAX® W8 (γ-cyclodextrin). In some embodiments, the cyclodextrin isCAVAMAX® W6 (α-cyclodextrin).

SEDDS are isotropic mixtures of oils, surfactants, solvents andco-solvents/surfactants, that can be used to improve the oral absorptionof highly lipophilic drug compounds. See, e.g., Tarate, B. et al.,Recent Patents on Drug Delivery & Formulation (2014) Vol. 8.

In some embodiments, the poly(ethylene glycol) molecule is a linearpolymer. The molecular weight of the linear chain PEG may be betweenabout 1,000 Da and about 100,000 Da. For example, a linear chain PEGused herein can have a molecular weight of about 100,000 Da, 95,000 Da,90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da,25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, or 1,000 Da. Insome embodiments, the molecular weight of the linear chain PEG isbetween about 1,000 Da and about 50,000 Da. In some embodiments, themolecular weight of the linear chain PEG is between about 1,000 Da andabout 40,000 Da. In some embodiments, the molecular weight of the linearchain PEG is between about 5,000 Da and about 40,000 Da. In someembodiments, the molecular weight of the linear chain PEG is betweenabout 5,000 Da and about 20,000 Da.

In some embodiments, the poly(ethylene glycol) molecule is a branchedpolymer. The molecular weight of the branched chain PEG may be betweenabout 1,000 Da and about 100,000 Da. For example, a branched chain PEGused herein can have a molecular weight of about 100,000 Da, 95,000 Da,90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da,25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, and 1,000 Da. Insome embodiments, the molecular weight of the branched chain PEG isbetween about 1,000 Da and about 50,000 Da. In some embodiments, themolecular weight of the branched chain PEG is between about 1,000 Da andabout 40,000 Da. In some embodiments, the molecular weight of thebranched chain PEG is between about 5,000 Da and about 40,000 Da. Insome embodiments, the molecular weight of the branched chain PEG isbetween about 5,000 Da and about 20,000 Da.

In some embodiments, the solubilizing agent can be present in the liquidformulation in an amount of about 5 wt. % to about 35 wt. %, about 10wt. % to about 25 wt. %, about 10 wt. % to about 20 wt. %, or about 13wt. % to about 17 wt. %. For example, the solubilizing agent can bepresent at about 5 wt. %, 7 wt. %, 10 wt. %, 13 wt. %, 15 wt. %, 17 wt.%, 20 wt. %, 23 wt. %, 26 wt. %, 30 wt. % or about 35 wt. %. In someembodiments, the solubilizing agent is present in the liquid formulationin an amount of 15 wt. %.

A buffer can be added to the liquid formulation to adjust the pH of theformulation to a desired pH. In some embodiments, a buffer can be addedin an amount to adjust the pH of the formulation to a pH of about 2 toabout 7, about 2.5 to about 5.5, or about 3 to about 4. For example, abuffer can be added in an amount to adjust the pH of the formulation toa pH of about 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, or about 7.0. Insome embodiments, a buffer can be added in an amount to adjust the pH ofthe formulation to a pH of about 3.5. In some embodiments, the bufferincludes a citrate buffer, a lactate buffer, a phosphate buffer, amaleate buffer, a tartrate buffer, a suucinate buffer, an acetatebuffer, or a combination thereof. In some embodiments, the bufferincludes lithium lactate, sodium lactate, potassium lactate, calciumlactate, lithium phosphate, sodium phosphate, potassium phosphate,calcium phosphate, lithium maleate, sodium maleate, potassium maleate,calcium maleate, lithium tartrate, sodium tartrate, potassium tartrate,calcium tartrate, lithium succinate, sodium succinate, potassiumsuccinate, calcium succinate, lithium acetate, sodium acetate, potassiumacetate, calcium acetate, or combinations thereof. In some embodiments,the buffer is a citrate buffer. For example, the citrate buffer caninclude at least one of lithium citrate monohydrate, sodium citratemonohydrate, potassium citrate monohydrate, calcium citrate monohydrate,lithium citrate dihydrate, sodium citrate dihydrate, potassium citratedihydrate, calcium citrate dihydrate, lithium citrate trihydrate, sodiumcitrate trihydrate, potassium citrate trihydrate, calcium citratetrihydrate, lithium citrate tetrahydrate, sodium citrate tetrahydrate,potassium citrate tetrahydrate, calcium citrate tetrahydrate, lithiumcitrate pentahydrate, sodium citrate pentahydrate, potassium citratepentahydrate, calcium citrate pentahydrate, lithium citrate hexahydrate,sodium citrate hexahydrate, potassium citrate hexahydrate, calciumcitrate hexahydrate, lithium citrate heptahydrate, sodium citrateheptahydrate, potassium citrate heptahydrate, calcium citrateheptahydrate, or mixtures thereof. The buffer can include sodium citratemonohydrate, potassium citrate monohydrate, calcium citrate monohydrate,sodium citrate dihydrate, potassium citrate dihydrate, calcium citratedihydrate, sodium citrate trihydrate, potassium citrate trihydrate,calcium citrate trihydrate, sodium citrate tetrahydrate, potassiumcitrate tetrahydrate, calcium citrate tetrahydrate, sodium citratepentahydrate, potassium citrate pentahydrate, calcium citratepentahydrate, sodium citrate hexahydrate, potassium citrate hexahydrate,calcium citrate hexahydrate, sodium citrate heptahydrate, potassiumcitrate heptahydrate, or calcium citrate heptahydrate. In someembodiments, the buffer includes sodium citrate dihydrate.

In some embodiments, the buffer is present in the liquid formulation inan amount of about 0.1 wt. % to about 5 wt. %, about 0.3 wt. % to about4 wt. %, about 0.5 wt. % to about 3.5 wt. %, about 0.6 wt. % to about 3wt. %, 0.7 wt. % to about 2.5 wt. %, about 0.7 wt. % to about 2.0 wt. %,or about 0.7 wt. % to about 1.5 wt. %. For example, the buffer can bepresent in the liquid formulation in an amount of about 0.1 wt. %, 0.3wt. %, 0.5 wt. %, 0.7 wt. %, 0.9 wt. %, 1.1 wt. %, 1.5 wt. %, 2.0 wt. %,2.5 wt. %, 3.0 wt. %, 3.5 wt. %, 4.0 wt. %, or about 5 wt. %. In someembodiments, the buffer is present in the liquid formulation in anamount of about 0.9 wt. %.

The pH of the liquid formulation can be adjusted to a desired pH. Insome embodiments, the pH of the formulation can be adjusted to a pH ofabout 2 to about 7, about 2.5 to about 5.5, or about 3 to about 4. Forexample, the pH of the formulation can be adjusted to a pH of about 2.0,2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, or about 7.0. In some embodiments,the pH of the formulation is adjusted to a pH of about 3.5. In some suchembodiments, where the pH of the liquid formulation is adjusted to adesired pH, the liquid formulation includes a base. In some embodiments,the base is selected from a citrate, a lactate, a phosphate, a maleate,a tartrate, a succinate, an acetate, a carbonate, a hydroxide, or acombination thereof. In some embodiments, the base includes lithiumlactate, sodium lactate, potassium lactate, calcium lactate, lithiumphosphate, sodium phosphate, potassium phosphate, calcium phosphate,lithium maleate, sodium maleate, potassium maleate, calcium maleate,lithium tartrate, sodium tartrate, potassium tartrate, calcium tartrate,lithium succinate, sodium succinate, potassium succinate, calciumsuccinate, lithium acetate, sodium acetate, potassium acetate, calciumacetate, sodium carbonate, potassium carbonate, calcium carbonate,sodium bicarbonate, potassium bicarbonate, calcium bicarbonate, sodiumhydroxide, potassium hydroxide, calcium hydroxide, or combinationsthereof. In some embodiments, the base includes a citrate. For example,the citrate can include at least one of lithium citrate monohydrate,sodium citrate monohydrate, potassium citrate monohydrate, calciumcitrate monohydrate, lithium citrate dihydrate, sodium citratedihydrate, potassium citrate dihydrate, calcium citrate dihydrate,lithium citrate trihydrate, sodium citrate trihydrate, potassium citratetrihydrate, calcium citrate trihydrate, lithium citrate tetrahydrate,sodium citrate tetrahydrate, potassium citrate tetrahydrate, calciumcitrate tetrahydrate, lithium citrate pentahydrate, sodium citratepentahydrate, potassium citrate pentahydrate, calcium citratepentahydrate, lithium citrate hexahydrate, sodium citrate hexahydrate,potassium citrate hexahydrate, calcium citrate hexahydrate, lithiumcitrate heptahydrate, sodium citrate heptahydrate, potassium citrateheptahydrate, calcium citrate heptahydrate, or mixtures thereof. Thebase can include sodium citrate monohydrate, potassium citratemonohydrate, calcium citrate monohydrate, sodium citrate dihydrate,potassium citrate dihydrate, calcium citrate dihydrate, sodium citratetrihydrate, potassium citrate trihydrate, calcium citrate trihydrate,sodium citrate tetrahydrate, potassium citrate tetrahydrate, calciumcitrate tetrahydrate, sodium citrate pentahydrate, potassium citratepentahydrate, calcium citrate pentahydrate, sodium citrate hexahydrate,potassium citrate hexahydrate, calcium citrate hexahydrate, sodiumcitrate heptahydrate, potassium citrate heptahydrate, or calcium citrateheptahydrate. In some embodiments, the base includes sodium citratedihydrate.

In some embodiments, the base is present in the liquid formulation in anamount of about 0.1 wt. % to about 5 wt. %, about 0.3 wt. % to about 4wt. %, about 0.5 wt. % to about 3.5 wt. %, about 0.6 wt. % to about 3wt. %, 0.7 wt. % to about 2.5 wt. %, about 0.7 wt. % to about 2.0 wt. %,or about 0.7 wt. % to about 1.5 wt. %. For example, the base can bepresent in the liquid formulation in an amount of about 0.1 wt. %, 0.3wt. %, 0.5 wt. %, 0.7 wt. %, 0.9 wt. %, 1.1 wt. %, 1.5 wt. %, 2.0 wt. %,2.5 wt. %, 3.0 wt. %, 3.5 wt. %, 4.0 wt. %, or about 5 wt. %. In someembodiments, the base is present in the liquid formulation in an amountof about 0.9 wt. %. For example, the citrate is present in the liquidformulation in an amount of about 0.1 wt. % to about 5 wt. %, about 0.3wt. % to about 4 wt. %, about 0.5 wt. % to about 3.5 wt. %, about 0.6wt. % to about 3 wt. %, 0.7 wt. % to about 2.5 wt. %, about 0.7 wt. % toabout 2.0 wt. %, or about 0.7 wt. % to about 1.5 wt. %. In someembodiments, the citrate can be present in the liquid formulation in anamount of about 0.1 wt. %, 0.3 wt. %, 0.5 wt. %, 0.7 wt. %, 0.9 wt. %,1.1 wt. %, 1.5 wt. %, 2.0 wt. %, 2.5 wt. %, 3.0 wt. %, 3.5 wt. %, 4.0wt. %, or about 5 wt. %. For example, the citrate is present in theliquid formulation in an amount of about 0.9 wt. %.

The liquid formulation can have a pH of about 2 to about 8, about 2.5 toabout 6, about 3 to about 4, or about 3 to about 4. For example, theliquid formulation can have a pH of about 2, 2.5, 3.0, 3.5, 4.0, 4.5, orabout 5. In some embodiments, the formulation can have a pH of about3.5.

A sweetener can be added to the liquid formulation to make it lessbitter or palatable, or both. Sweeteners suitable for inclusion in theformulation can include, both natural and artificial sweeteners. In someembodiments, the sweetener is an artificial sweetener and can includeintense or high-intensity sweeteners. Intense sweeteners are commonlyused as sugar substitutes or sugar alternatives as they are many timessweeter than sugar but contribute only a few to no calories when addedto food. Exemplary intense sweeteners include sorbitol, sucrose,saccharins such as sodium saccharin, cyclamates such as sodiumcyclamates, aspartame, sucralose, thaumatin, and acesulfam K. In someembodiments, the sweetener is a natural sugar. For example, sugars suchas monosaccharides, disaccharides and polysaccharides can be used in theliquid formulations provided herein. The sugars can include xylose,ribose, glucose, mannose, galactose, fructose, dextrose, sucrose,maltose, partially hydrolyzed starch or corn syrup, and sugar alcoholssuch as sorbitol, xylitol, mannitol, glycerin, and combination thereof.In some embodiments, the liquid formulation further comprises asweetener. The sweetener can include a sugar. For example, the sweetenercan include sucrose. For example, the sweetener can be ORA-SWEET®, asweetener that includes purified water, sucrose, glycerin, sorbitol, andflavoring; is buffered with citric acid and sodium phosphate; and ispreserved with methylparaben and potassium sorbate. The sweetener canalso include an intense sweetener. The intense sweetener can includesucralose. For example, the sweetener can be ORA-SWEET SF®, a sugar freesweetener that includes purified water, glycerin, sorbitol, sodiumsaccharin, xanthan gum, and flavoring; is buffered with citric acid andsodium citrate; and is preserved with methylparaben (0.03%), potassiumsorbate (0.1%), and propylparaben (0.008%).

In some embodiments, the sweetener includes one or more of sucrose,glycerin, sorbitol, and flavoring. In some such embodiments, thesweetener further includes citric acid and sodium phosphate. In somesuch embodiments, the sweetener can include a preservative, such asmethylparaben and potassium sorbate. For example, the sweetener includessucrose, glycerin, sorbitol, flavoring, citric acid, sodium phosphate,methylparaben, and potassium sorbate. In some embodiments, the sweetenerincludes one or more of glycerin, sorbitol, sodium saccharin, xanthangum, and flavoring. In some such embodiments, the sweetener furtherincludes citric acid and sodium citrate. In some such embodiments, thesweetener includes a preservative, such as methylparaben, potassiumsorbate, and propylparaben. For example, the sweetener can includeglycerin, sorbitol, sodium saccharin, xanthan gum, flavoring, citricacid and sodium citrate, methylparaben (0.03%), potassium sorbate(0.1%), and propylparaben (0.008%).

In some embodiments, the sweetener is present in the liquid formulationin an amount of about 30 wt. % to about 70 wt. %, about 35 wt. % toabout 65 wt. %, about 40 wt. % to about 60 wt. %, or about 45 wt. % toabout 55 wt. %. For example, the sweetener can be present in the liquidformulation in an amount of about 30 wt. %, 35 wt. %, 40 wt. %, 45 wt.%, 50 wt. %, 55 wt. %, 60 wt. %, 65 wt. %, or about 70 wt. %. In someembodiments, the sweetener is present in the liquid formulation in anamount of about 50 wt. %.

In some embodiments, the liquid formulation further comprises abitterness masking agent. The bitterness masking agent can include231a12 natural masking type flavor (Abelei®), 231a39 natural bitternessmasking type flavor (Abelei®), bitterness masking flavor, nat (FONA®),and FINATECH Taste Modifier Flavor, Nat.

The bitterness masking agent can be present in the liquid formulation inan amount of about 0.01 wt. % to about 2 wt. %, about 0.1 wt. % to about1.0 wt. %, or about 0.2 wt. % to about 0.5 wt. %. For example, thebitterness masking agent can be present in the liquid formulation in anamount of about 0.01 wt. %, 0.1 wt. %, 0.2 wt. %, 0.3 wt. %, 0.4 wt. %,0.5 wt. %, 0.7 wt. %, 1.0 wt. %, 1.5 wt. %, or 2.0 wt. %. In someembodiments, the bitterness masking agent is present in the liquidformulation in an amount of about 0.4 wt. %.

A flavoring agent can be included in the liquid formulation so that thefinal formulation has a substantially non-bitter and palatable taste.The flavoring agent can include at least one of a natural flavoringagent, a natural fruit flavoring agent, an artificial flavoring agent,an artificial fruit flavoring agent, flavor enhancers, or mixturesthereof. Exemplary flavoring agents can be found, for example in US CFR21 §172.515 (Apr. 1, 2015), which is incorporated by reference in itsentirety. For example, cinnamon, raspberry, orange, maple, butterscotch,glycyrrhiza (licorice) syrup, fruit, berry, vanilla, acacia syrup, coca,chocolate-mint, wild cherry, walnut, eriodictyon, bubblegum, grapefruit,lime, marshmellow, gurana, coffee, peach, lemon, fennel, apricot, honey,mint, wintergreen, and cherry. In some embodiments, the flavoring agentcan include a FONATECH® natural taste modifier flavoring agent. Theflavoring agent can be present in the liquid formulation in an amount ofabout 0.01 wt. % to about 2 wt. %, about 0.01 wt. % to about 0.1 wt. %,or about 0.2 wt. % to about 0.5 wt. %. For example, the flavoring agentcan be present in an amount of about 0.01 wt. %, 0.1 wt. %, 0.2 wt. %,0.3 wt. %, 0.4 wt. %, 0.5 wt. %, 0.7 wt. %, 1.0 wt. %, 1.5 wt. %, or 2.0wt. %. In some embodiments, the flavoring agent can be present in theliquid formulation in an amount of about 0.5 wt. %.

The liquid formulation can also include a coloring agent.

The liquid formulations provided herein can be prepared from acrystalline form of the compound of formula (I). The crystalline formcan the formula (I-HS):

Also provided herein is a liquid formulation including(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehaving the formula (I):

a pharmaceutically acceptable salt thereof, or a combination thereof, asolubilizing agent and a buffer. In some embodiments, the formulationhas a pH of about 2.5 to about 5.5. In some embodiments, the compound offormula (I) has a concentration of about 15 mg/mL to about 35 mg/mL. Insome embodiments, the formulation has a pH of about 3 to about 4 and thecompound of formula (I), or a pharmaceutically acceptable salt thereof,or a combination thereof, is present at a concentration of about 15mg/mL to about 35 mg/mL in the liquid formulation. The buffer caninclude sodium citrate dihydrate.

Also provided herein is a liquid formulation including(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehaving the formula (I):

a pharmaceutically acceptable salt thereof, or a combination thereof, asolubilizing agent and a base. In some embodiments, the formulation hasa pH of about 2.5 to about 5.5. In some embodiments, the compound offormula (I) has a concentration of about 15 mg/mL to about 35 mg/mL. Insome embodiments, the formulation has a pH of about 3 to about 4 and thecompound of formula (I), or a pharmaceutically acceptable salt thereof,or a combination thereof, is present at a concentration of about 15mg/mL to about 35 mg/mL in the liquid formulation. The base can includesodium citrate dihydrate.

Also provided herein is a liquid formulation including(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehaving the formula (I):

a pharmaceutically acceptable salt thereof, or a combination thereof, asolubilizing agent, a buffer, a sweetener, a bitterness masking agent,and a flavoring agent. In some embodiments, the formulation has a pH ofabout 3 to about 4 and the compound of formula (I), or apharmaceutically acceptable salt thereof, or a combination thereof, ispresent at a concentration of about 15 mg/mL to about 35 mg/mL in theliquid formulation. In some embodiments, the buffer includes sodiumcitrate dihydrate. In some embodiments, the sweetener includes sucrose.

Also provided herein is a liquid formulation including(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehaving the formula (I):

a pharmaceutically acceptable salt thereof, or a combination thereof, asolubilizing agent, a base, a sweetener, a bitterness masking agent, anda flavoring agent. In some embodiments, the formulation has a pH ofabout 3 to about 4 and the compound of formula (I), or apharmaceutically acceptable salt thereof, or a combination thereof, ispresent at a concentration of about 15 mg/mL to about 35 mg/mL in theliquid formulation. In some embodiments, the base includes sodiumcitrate dihydrate. In some embodiments, the sweetener includes sucrose.

Also provided herein is a liquid formulation including(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehaving the formula (I):

a pharmaceutically acceptable salt thereof, or a combination thereof, asolubilizing agent, a buffer, a sweetener, a bitterness masking agent,and flavoring agent, wherein the formulation has a pH of about 3 toabout 4.

Also provided herein is a liquid formulation including(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehaving the formula (I):

a pharmaceutically acceptable salt thereof, or a combination thereof, asolubilizing agent, a base, a sweetener, a bitterness masking agent, andflavoring agent, wherein the formulation has a pH of about 3 to about 4.

Also provided herein is a liquid formulation including(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehaving the formula (I):

a pharmaceutically acceptable salt thereof, or a combination thereof, asolubilizing agent, a buffer, a sweetener, a bitterness masking agent,and a flavoring agent, wherein the compound of formula (I) has aconcentration of about 15 mg/mL to about 35 mg/mL in the liquidformulation.

Also provided herein is a liquid formulation including(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehaving the formula (I):

a pharmaceutically acceptable salt thereof, or a combination thereof, asolubilizing agent, a base, a sweetener, a bitterness masking agent, anda flavoring agent, wherein the compound of formula (I) has aconcentration of about 15 mg/mL to about 35 mg/mL in the liquidformulation.

Also provided herein is a liquid formulation including:

(a)(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehaving the formula (I):

a pharmaceutically acceptable salt thereof, or a combination thereof;(b) a solubilizing agent present in an amount of about 5 wt. % to about35 wt. %; and(c) a buffer present in an amount of about 0.1 wt. % to about 5 wt. %.In some embodiments, the buffer comprises sodium citrate dehydrate. Insome embodiments, the formulation also includes a sweetener present inan amount of about 30 wt. % to about 70 wt. %. In some embodiments, thesweetener comprises sucrose. In some embodiments, the formulation alsoincludes a bitterness masking agent present in an amount of about 0.2wt. % to about 0.5 wt. %. In some embodiments, the formulation alsoincludes a flavoring agent present in an amount of about 0.01 wt. % toabout 2 wt. %. In some embodiments, the formulation has a pH of about 3to about 4. In some embodiments, the compound of formula (I) has aconcentration of about 20 mg/mL to about 30 mg/mL in the liquidformulation.

Also provided herein is a liquid formulation including:

(a)(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehaving the formula (I):

a pharmaceutically acceptable salt thereof, or a combination thereof;(b) a solubilizing agent present in an amount of about 5 wt. % to about35 wt. %; and(c) a base present in an amount of about 0.1 wt. % to about 5 wt. %. Insome embodiments, the base comprises sodium citrate dehydrate. In someembodiments, the formulation also includes a sweetener present in anamount of about 30 wt. % to about 70 wt. %. In some embodiments, thesweetener comprises sucrose. In some embodiments, the formulation alsoincludes a bitterness masking agent present in an amount of about 0.2wt. % to about 0.5 wt. %. In some embodiments, the formulation alsoincludes a flavoring agent present in an amount of about 0.01 wt. % toabout 2 wt. %. In some embodiments, the formulation has a pH of about 3to about 4. In some embodiments, the compound of formula (I) has aconcentration of about 20 mg/mL to about 30 mg/mL in the liquidformulation.

Also provided herein is a liquid formulation including:

(a)(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehaving the formula (I):

a pharmaceutically acceptable salt thereof, or a combination thereof;(b) a solubilizing agent (e.g., a cyclodextrin such as ahydroxypropyl-β-cyclodextrin) present in an amount of about 5 wt. % toabout 35 wt. %; and(c) a buffer (e.g., a citrate buffer such as sodium citrate) present inan amount of about 0.1 wt. % to about 5 wt. %;(d) a sweetener (e.g., a sweetener comprising sucrose or an intensesweetener) present in an amount of about 30 wt. % to about 70 wt. %;(e) a bitterness masking agent present in an amount of about 0.2 wt. %to about 0.5 wt. %.; and(f) a flavoring agent present in an amount of about 0.01 wt. % to about2 wt. %. In some embodiments, the formulation has a pH of about 3 toabout 4. In some embodiments, the compound of formula (I) has aconcentration of about 20 mg/mL to about 30 mg/mL in the liquidformulation.

Also provided herein is a liquid formulation including:

(a)(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehaving the formula (I):

a pharmaceutically acceptable salt thereof, or a combination thereof;(b) a solubilizing agent (e.g., a cyclodextrin such as ahydroxypropyl-3-cyclodextrin) present in an amount of about 5 wt. % toabout 35 wt. %; and(c) a base (e.g., a citrate such as sodium citrate) present in an amountof about 0.1 wt. % to about 5 wt. %;(d) a sweetener (e.g., a sweetener comprising sucrose or an intensesweetener) present in an amount of about 30 wt. % to about 70 wt. %;(e) a bitterness masking agent present in an amount of about 0.2 wt. %to about 0.5 wt. %.; and(f) a flavoring agent present in an amount of about 0.01 wt. % to about2 wt. %. In some embodiments, the formulation has a pH of about 3 toabout 4. In some embodiments, the compound of formula (I) has aconcentration of about 20 mg/mL to about 30 mg/mL in the liquidformulation.

Also provided herein is a liquid formulation including:

(a)(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehaving the formula (I):

a pharmaceutically acceptable salt thereof, or a combination thereof;(b) hydroxypropyl-β-cyclodextrin present in an amount of about 5 wt. %to about 35 wt. %; and(c) a sodium citrate present in an amount of about 0.1 wt. % to about 5wt. %;(d) a sucrose or an intense sweetener present in an amount of about 30wt. % to about 70 wt. %;(e) a bitterness masking agent present in an amount of about 0.2 wt. %to about 0.5 wt. %.; and(f) a flavoring agent present in an amount of about 0.01 wt. % to about2 wt. %. In some embodiments, the formulation has a pH of about 3 toabout 4. In some embodiments, the compound of formula (I) has aconcentration of about 20 mg/mL to about 30 mg/mL in the liquidformulation.

Also provided herein is a liquid formulation including:

(a)(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehaving the formula (I):

a pharmaceutically acceptable salt thereof, or a combination thereof;(b) hydroxypropyl-β-cyclodextrin present in an amount of about 5 wt. %to about 35 wt. %; and(c) sodium citrate dihydrate present in an amount of about 0.1 wt. % toabout 5 wt. %;(d) a sucrose or an intense sweetener present in an amount of about 30wt. % to about 70 wt. %;(e) a bitterness masking agent present in an amount of about 0.2 wt. %to about 0.5 wt. %.; and(f) a flavoring agent present in an amount of about 0.01 wt. % to about2 wt. %. In some embodiments, the formulation has a pH of about 3 toabout 4. In some embodiments, the compound of formula (I) has aconcentration of about 20 mg/mL to about 30 mg/mL in the liquidformulation.

In some embodiments, the liquid formulation is prepared from apharmaceutically acceptable salt of the compound of formula (I). Forexample, the pharmaceutically acceptable salt is a hydrogen sulfatesalt. In some embodiments, the liquid formulation is prepared from acrystalline form of the compound of formula (I). For example, thecrystalline form of the compound of formula (I) can have the formula(I-HS):

In some embodiments, crystalline form (I-HS) is characterized by havingXRPD diffraction peaks (2θ degrees) at 18.4±0.2, 20.7±0.2, 23.1±0.2, and24.0±0.2. In some embodiments, crystalline form (I-HS) is characterizedby having XRPD diffraction peaks (20 degrees) at 10.7±0.2, 18.4±0.2,20.7±0.2, 23.1±0.2, and 24.0±0.2. In some embodiments, crystalline form(I-HS) is characterized by having XRPD diffraction peaks (2θ degrees) at10.7±0.2, 18.4±0.2, 19.2±0.2, 20.2±0.2, 20.7±0.2, 21.5±0.2, 23.1±0.2,and 24.0±0.2. In some embodiments, crystalline form (I-HS) ischaracterized by having XRPD diffraction peaks (20 degrees) at 10.7±0.2,15.3±0.2, 16.5±0.2, 18.4±0.2, 19.2±0.2, 19.9±0.2, 20.2±0.2, 20.7±0.2,21.5±0.2, 22.1±0.2, 23.1±0.2, 24.0±0.2, 24.4±0.2, 25.6±0.2, 26.5±0.2,27.6±0.2, 28.2±0.2, 28.7±0.2, 30.8±0.2, and 38.5±0.2.

In some embodiments, the crystalline form (I-HS) has XRPD patternsubstantially as shown in FIG. 1 or FIG. 8.

In some embodiments, the crystalline form exhibits an onset to maximumof about 193 to about 205° Celsius, as measured by differential scanningcalorimetry. In some embodiments, the crystalline form (I-HS) exhibits aheat of melting of about 2.415 mW, as measured by differential scanningcalorimetry.

Also provided herein is a method of treating cancer in a patient in needthereof. The method includes administering to the patient atherapeutically effective amount of a liquid formulation providedherein.

In some embodiments, the cancer results in dysphagia or difficultyswallowing. For example, the cancer can be a head and neck cancer, amouth cancer, a throat cancer, or an esophageal cancer. In someembodiments, a patient having cancer develops difficulty swallowing dueto one or more of fibrosis in the throat, esophagus, or mouth;infections of the mouth or esophagus (e.g., from radiation therapy orchemotherapy), swelling or narrowing of the throat or esophagus (e.g.,from radiation therapy or surgery); physical changes to the mouth, jaws,throat, or esophagus from surgery; muscositis, which is soreness, painor inflammation in the throat, esophagus, or mouth; xerostomia, commonlyreferred to as dry mouth (e.g., from radiation therapy or chemotherapy).

In some embodiments, the patient is an infant, a child, an adolescent,or an elderly patient.

In some embodiments, the patient has a dysphagia. The dysphagia can bean oropharyngeal dysphagia. Oropharyngeal dysphagia can arise due tocancer (e.g., certain cancers and some cancer treatments, such asradiation, can cause difficulty swallowing), neurological disorders(e.g., certain disorders, such as multiple sclerosis, muscular dystrophyand Parkinson's disease, can cause dysphagia), neurological damage(e.g., sudden neurological damage, such as from a stroke or brain orspinal cord injury, that effects one's ability to swallow), andpharyngeal diverticula.

In some embodiments, the patient has a neurological disorders (e.g.,certain disorders, such as multiple sclerosis, muscular dystrophy andParkinson's disease, can cause dysphagia), neurological damage (e.g.,sudden neurological damage, such as from a stroke or brain or spinalcord injury, that effects one's ability to swallow), and pharyngealdiverticula.

Also provided herein is a method of treating cancer in a patient in needthereof with dysphagia (e.g., difficulty swallowing). The methodincludes identifying a patient in need thereof with dysphagia. Themethod further includes administering to the patient a therapeuticallyeffective amount of a liquid formulation described herein.

In some embodiments, the dysphagia is an oropharyngeal dysphagia.

Also provided herein is a method of treating cancer in a patient in needthereof with dysphagia. The method includes identifying a patient inneed thereof with dysphagia. The method further includes determining ifthe cancer is mediated by a Trk kinase. If the cancer is determined tobe mediated by a Trk kinase, administering to the patient atherapeutically effective amount of a liquid formulation describedherein.

In some embodiments, the dysphagia is an oropharyngeal dysphagia.Oropharyngeal dysphagia can arise due to cancer (e.g., certain cancersand some cancer treatments, such as radiation, can cause difficultyswallowing), neurological disorders (e.g., certain disorders, such asmultiple sclerosis, muscular dystrophy and Parkinson's disease, cancause dysphagia), neurological damage (e.g., sudden neurological damage,such as from a stroke or brain or spinal cord injury, that effects one'sability to swallow), and pharyngeal diverticula.

Definitions

Where the compound disclosed herein has at least one chiral center, thecompounds may accordingly exist as enantiomers. Where the compoundspossess two chiral centers, the compounds may additionally exist asdiastereomers. That is, the compound of formula (I), in addition tohaving the desired configuration designated by the nomenclature“(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehydrogen sulfate” (hereinafter referred to as the (S,R) isomer), it mayalso be present in minor amounts as the isomer(R)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehydrogen sulfate (hereinafter referred to as the (R,R) isomer) and/ormay also be present in minor amounts as the(S)—N-(5-((S)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehydrogen sulfate (hereinafter referred to as the (S,S) isomer), and/ormay be present in minor amounts as the isomer(R)—N-(5-((S)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehydrogen sulfate” (hereinafter referred to as the (R,S) isomer). It isto be understood that all such isomers and mixtures thereof areencompassed within the scope of the present invention. Preferably,wherein the compound is present as the (S,R) isomer, the (S,R) isomer ispresent at an excess of greater than or equal to about 80%, morepreferably at an excess of greater than or equal to about 90%, morepreferably still at an excess of greater than or equal to about 95%,more preferably still at an excess of greater than or equal to about98%, more preferably at an excess of greater than or equal to about 99%.

As used herein, unless otherwise noted, the term “isolated form” shallmean that the compound is present in a form which is separate from anysolid mixture with another compound(s), solvent system or biologicalenvironment. In some embodiments, the crystalline form (I-HS) is presentas an isolated form.

As used herein, unless otherwise noted, the term “substantially pureform” shall mean that the mole percent of impurities in the isolatedcompound or crystalline form is less than about 5 mole percent,preferably less than about 2 mole percent, more preferably, less thanabout 0.5 mole percent, most preferably, less than about 0.1 molepercent. In some embodiments, the crystalline form (I-HS) is present asa substantially pure form.

As used herein, unless otherwise noted, the term “substantially free ofother amorphous, polymorph or crystalline form(s)” when used todescribed crystalline form (I-HS) shall mean that mole percent of otheramorphous, polymorph or crystalline form(s) of the isolated base ofcrystalline form (I-HS) is less than about 5 mole percent, preferablyless than about 2 mole percent, more preferably, less than about 0.5mole percent, most preferably less than about 0.1 mole percent. In someembodiments, the crystalline form (I-HS) is present as a formsubstantially free of other amorphous, polymorph or crystalline form(s).

The terms “polymorph” and “polymorphic form” refer to differentcrystalline forms of a single compound. That is, polymorphs are distinctsolids sharing the same molecular formula, yet each polymorph may havedistinct solid state physical properties. Therefore, a single compoundmay give rise to a variety of polymorphic forms where each form hasdifferent and distinct solid state physical properties, such asdifferent solubility profiles, dissolution rates, melting pointtemperatures, flowability, and/or different X-ray diffraction peaks. Thedifferences in physical properties may affect pharmaceutical parameterssuch as storage stability, compressibility and density (which can beimportant in formulation and product manufacturing), and dissolutionrate (which can be an important factor in bioavailability). Techniquesfor characterizing polymorphic forms include, but are not limited to,X-ray powder diffractometry (XRPD), differential scanning calorimetry(DSC), thermal gravimetric analysis (TGA), single-crystal X-raydiffractometry (XRD), vibrational spectroscopy, e.g., infrared (IR) andRaman spectroscopy, solid-state and solution nuclear magnetic resonance(NMR) spectroscopy, optical microscopy, hot stage optical microscopy,scanning electron microscopy (SEM), electron crystallography andquantitative analysis, particle size analysis (PSA), surface areaanalysis, solubility measurements, dissolution measurements, elementalanalysis and Karl Fischer analysis.

The term “amorphous” means a solid in a solid state that is anon-crystalline state. Amorphous solids are disordered arrangements ofmolecules and therefore possess no distinguishable crystal lattice orunit cell and consequently have no definable long range ordering. Thesolid state form of a solid may be determined by polarized lightmicroscopy, X-ray powder diffraction (“XRPD”), differential scanningcalorimetry (“DSC”), or other standard techniques known to those ofskill in the art.

As used herein, the term “pharmaceutically acceptable salts” refers tosalts that retain the desired biological activity of the subjectcompound and exhibit minimal undesired toxicological effects. Thesepharmaceutically acceptable salts may be prepared in situ during thefinal isolation and purification of the compound, or by separatelyreacting the purified compound in its free acid or free base form with asuitable base or acid, respectively. In some embodiments,pharmaceutically acceptable salts may be preferred over the respectivefree base or free acid because such salts impart greater stability orsolubility to the molecule thereby facilitating formulation into adosage form. Basic compounds are generally capable of formingpharmaceutically acceptable acid addition salts by treatment with asuitable acid.

Suitable acids include pharmaceutically acceptable inorganic acids andpharmaceutically acceptable organic acids. Representativepharmaceutically acceptable acid addition salts include hydrochloride,hydrobromide, nitrate, methylnitrate, sulfate, bisulfate, sulfamate,phosphate, acetate, hydroxyacetate, phenylacetate, propionate, butyrate,isobutyrate, valerate, maleate, hydroxymaleate, acrylate, fumarate,malate, tartrate, citrate, salicylate, p-aminosalicyclate, glycollate,lactate, heptanoate, phthalate, oxalate, succinate, benzoate,o-acetoxybenzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate,hydroxybenzoate, methoxybenzoate, mandelate, tannate, formate, stearate,ascorbate, palmitate, oleate, pyruvate, pamoate, malonate, laurate,glutarate, glutamate, estolate, methanesulfonate (mesylate),ethanesulfonate (esylate), 2-hydroxyethanesulfonate, benzenesulfonate(besylate), p-aminobenzenesulfonate, p-toluenesulfonate (tosylate),napthalene-2-sulfonate, Ethanedisulfonate, and 2,5-dihydroxybenzoate.

As used herein, unless otherwise noted, the terms “treating,”“treatment,” and the like, shall include the management and care of asubject or patient (preferably mammal, more preferably human) for thepurpose of combating a disease, condition, or disorder and includes theadministration of a disclosed compound to alleviate the symptoms orcomplications, or reduce the rate of progression of the disease,condition, or disorder.

As used herein, unless otherwise noted, the term “prevention” shallinclude (a) reduction in the frequency of one or more symptoms; (b)reduction in the severity of one or more symptoms; (c) the delay oravoidance of the development of additional symptoms; and/or (d) delay oravoidance of the development of the disorder or condition.

As used herein, the term “Trk-associated cancer” shall be defined toinclude cancers associated with or having dysregulation of a NTRK gene,a Trk protein, or expression or activity, or level of the same (e.g.,any of types of dysregulation of a NTRK gene, a Trk protein, orexpression or activity, or level of the same, described herein).Non-limiting examples of a Trk-associated cancer are described herein.

The term “subject” as used herein, refers to an animal, preferably amammal, most preferably a human, who has been the object of treatment,observation or experiment. In some embodiments, the subject hasexperienced and/or exhibited at least one symptom of the disease ordisorder to be treated and/or prevented. In some embodiments, a patientis a pediatric patient (i.e. a patient under the age of 21 years at thetime of diagnosis or treatment).

The term “pediatric” can be further divided into various subpopulationsincluding: neonates (from birth through the first 28 days of life);infants (29 days of age to less than two years of age); children (twoyears of age to less than 12 years of age); and adolescents (12 years ofage through 21 years of age (up to, but not including, the twenty-secondbirthday)). Berhman R E, Kliegman R, Arvin A M, Nelson W E. NelsonTextbook of Pediatrics, 15th Ed. Philadelphia: W.B. Saunders Company,1996; Rudolph A M, et al. Rudolph's Pediatrics, 21st Ed. New York:McGraw-Hill, 2002; and Avery M D, First L R. Pediatric Medicine, 2nd Ed.Baltimore: Williams & Wilkins; 1994.

The term “Trk” or “Trk protein” includes any of the Trk proteinsdescribed herein (e.g., a TrkA, a TrkB, or a TrkC protein).

The term “NTRK gene” includes any of the NTRK genes described herein(e.g., a NTRK1, a NTRK2, or a NTRK3 gene).

The term “wildtype” or “wild-type” describes a nucleic acid (e.g., aNTRK gene or a Trk mRNA) or protein (e.g., a Trk protein) that is foundin a subject (e.g., a pediatric subject, e.g., an infant, child, oradolescent) that does not have a Trk-associated cancer (and optionallyalso does not have an increased risk of developing a Trk-associatedcancer or condition and/or is not suspected of having a Trk-associatedcancer or condition) or is found in a cell or tissue from a subject(e.g., a pediatric subject, e.g., an infant, child, or adolescent) thatdoes not have a Trk-associated cancer or condition (and optionally alsodoes not have an increased risk of developing a Trk-associated cancer orcondition and/or is not suspected of having a Trk-associated cancer orcondition).

The term “regulatory agency” is a country's agency for the approval ofthe medical use of pharmaceutical agents with the country. For example,a non-limiting example of a regulatory agency is the U.S. Food and DrugAdministration (FDA).

The phrase “dysregulation of a NTRK gene, a Trk protein, or expressionor activity, or level of the same” is a genetic mutation (e.g., a NTRKgene translocation that results in the expression of a fusion protein, adeletion in a NTRK gene that results in the expression of a Trk proteinthat includes a deletion of at least one amino acid as compared to thewild-type Trk protein, or a mutation in a NTRK gene that results in theexpression of a Trk protein with one or more point mutations, analternative spliced version of a Trk mRNA that results in a Trk proteinthat results in the deletion of at least one amino acid in the Trkprotein as compared to the wild-type Trk protein), or a NTRK geneduplication that results in overexpression of a Trk protein) oroverexpression of a NTRK gene in a cell, that results in a pathogenicincrease in the activity of a kinase domain of a Trk protein (e.g., aconstitutively active kinase domain of a Trk protein) in a cell. Forexample, a dysregulation of a NTRK gene, a Trk protein, or expression oractivity, or level of the same, can be a mutation in a NTRK1, NTRK2, orNTRK3 gene that encodes a Trk protein that is constitutively active orhas increased activity as compared to a protein encoded by a NTRK1,NTRK2, or NTRK3 gene that does not include the mutation. For example, adysregulation of a NTRK gene, a Trk protein, or expression or activity,or level of the same, can be the result of a gene translocation whichresults in the expression of a fusion protein that contains a firstportion of TrkA, TrkB, or TrkC that includes a functional kinase domain,and a second portion of a partner protein (i.e., that is not TrkA. TrkB,or TrkC). A gene encoding a fusion protein can include, e.g., thefollowing exons of a wild-type NTRK1 gene: exons 10-19, exons 12-19,exons 12-19, exons 13-19, exons 14-19, or exons 15-19. A gene encoding afusion protein can include, e.g., the following exons of a wild-typeNTRK2 gene: exons 12-21, exons 13-21, exons 15-21, exons 16-21, or exons17-21. A gene encoding a fusion protein can include, e.g., the followingexons of a wild-type NTRK3 gene: exons 17-22 or exons 16-22.Non-limiting examples of fusion proteins that are a result of a NTRKgene translocation are described in Table 10.

A dysregulation of a NTRK gene, a Trk protein, or expression oractivity, or level of the same, can, e.g., include a mutation(s) in aNTRK1, NTRK2, or NTRK3 gene that results in a TrkA, TrkB, or TrkCcontaining at least one (e.g., two, three, four, or five) pointmutations (e.g., one of more of the point mutations listed in Table XX).A dysregulation of a NTRK gene, a Trk protein, or expression oractivity, or level of the same, can be a mutation in a NTRK1, NTRK2, orNTRK3 gene that results in a deletion of one or more contiguous aminoacids (e.g., at least two, at least three, at least four, at least 5, atleast 6, at least 7, at least 8, at least 9, at least 10, at least 15,at least 20, at least 30, at least 40, at least 50, at least 60, atleast 70, at least 80, at least 90, at least 100, at least 110, at least120, at least 130, at least 140, at least 150, at least 160, at least170, at least 180, at least 190, at least 200, at least 210, at least220, at least 230, at least 240, at least 250, at least 260, at least270, at least 280, at least 290, at least 300, at least 310, at least320, at least 330, at least 340, at least 350, at least 360, at least370, at least 380, at least 390, or at least 400 amino acids) in theTrkA, TrkB, or TrkC protein (except for the deletion of amino acids inthe kinase domain of TrkA, TrkB, or TrkC that would result ininactivation of the kinase domain). In some examples, a dysregulation ofa NTRK gene, a Trk protein, or expression or activity, or level of thesame, can include an alternate spliced form of a Trk mRNA, e.g., aTrkAIII spliced variant or an alternative spliced form of a TrkA mRNAthat results in the production of a TrkA protein that lacks the aminoacids encoded by exon 10. In some examples, a dysregulation of a NTRKgene, a Trk protein, or expression or activity, or level of the same,includes an amplification of a NTRK gene (e.g., one, two, three, or fouradditional copies of the NTRK gene) that can result, e.g., in autocrineor overexpression of a NTRK gene in a cell. The term “overexpression” isa term of art and is used to an increased level of transcription of agene in a cell as compared to the level of transcription of the gene ina control cell (e.g., a non-cancerous cell of the same cell type).

The term “Trk-associated cancer or tumor” is a cancer that is associatedwith dysregulation of a NTRK gene, a Trk protein, or expression oractivity, or level of the same (e.g., a cancer that is associated withat least one example (e.g., two, three, four, or five examples) ofdysregulation of a NTRK gene, a Trk protein, or expression or activity,or level of the same, described herein).

The term “mammal” as used herein, refers to a warm-blooded animal thathas or is at risk of developing a disease described herein and includes,but is not limited to, guinea pigs, dogs, cats, rats, mice, hamsters,and primates, including humans.

The term “therapeutically effective amount” as used herein, means thatamount of active compound or pharmaceutical agent that elicits thebiological or medicinal response in a tissue system, animal or humanthat is being sought by a researcher, veterinarian, medical doctor orother clinician, which includes alleviation of the symptoms of thedisease or disorder being treated. In particular, a therapeuticallyeffective amount, when administered to a subject in need of suchtreatment, is sufficient to (i) treat or prevent a particular disease,condition, or disorder which can be treated with an inhibitor of TrkAand/or TrkB, (ii) attenuate, ameliorate, or eliminate one or moresymptoms of the particular disease, condition, or disorder, or (iii)prevent or delay the onset of one or more symptoms of the particulardisease, condition, or disorder described herein. The amount ofcrystalline form (I-HS) that will correspond to such a therapeuticallyeffective amount will vary depending upon factors such the diseasecondition and its severity, the identity (e.g., weight) of the mammal inneed of treatment, but can nevertheless be routinely determined by oneskilled in the art.

As used herein, the term “composition” is intended to encompass aproduct comprising the specified ingredients in the specified amounts,as well as any product which results, directly or indirectly, fromcombinations of the specified ingredients in the specified amounts.

To provide a more concise description, some of the quantitativeexpressions given herein are not qualified with the term “about.” It isunderstood that whether the term “about” is used explicitly or not,every quantity given herein is meant to refer to the actual given value,and it is also meant to refer to the approximation to such given valuethat would reasonably be inferred based on the ordinary skill in theart, including approximations due to the experimental and/or measurementconditions for such given value.

In some embodiments, the term “about” is used herein to meanapproximately, in the region of, roughly, or around. When the term“about” is used in conjunction with a numerical range, it modifies thatrange by extending the boundaries above and below the numerical valuesset forth. In general, the term “about” is used herein to modify anumerical value above and below the stated value by a variance of 10%.

The term “about” preceding one or more peak positions in an X-ray powderdiffraction pattern means that all of the peaks of the group which itprecedes are reported in terms of angular positions (two theta) with anallowable variability of ±0.3°. The variability of ±0.3° is intended tobe used when comparing two powder X-ray diffraction patterns. Inpractice, if a diffraction pattern peak from one pattern is assigned arange of angular positions (two theta) which is the measured peakposition ±0.3° and if those ranges of peak positions overlap, then thetwo peaks are considered to have the same angular position. For example,if a peak from one pattern is determined to have a position of 11.0°,for comparison purposes the allowable variability allows the peak to beassigned a position in the range of 10.7°-11.3°.

The term “about” preceding a value for DSC, TGA, TG, or DTA, which arereported as degrees Celsius, have an allowable variability of ±5° C.

To provide a more concise description, some of the quantitativeexpressions herein are recited as a range from about amount X to aboutamount Y. It is understood that wherein a range is recited, the range isnot limited to the recited upper and lower bounds, but rather includesthe full range from about amount X through about amount Y, or any rangetherein.

One skilled in the art will further recognize that human clinical trialsincluding first-in-human, dose ranging and efficacy trials, in healthypatients and/or those suffering from a given disorder, may be completedaccording to methods well known in the clinical and medical arts. Forexample, determining proper dosages for pediatric patients can bedetermined using known methods, including weight, age, and models suchas Simcyp® Pediatric Simulation modeling (CERTARA, Princeton, N.J.)which can be used to establish a pharmacokinetic approach for dosingthat takes into account patient age, ontogeny of the clearance pathwaysthat a compound of formula (I), a pharmaceutically acceptable saltthereof, or a combination thereof, and body surface area (BSA).

Acronyms found in the specification have the following meanings:

ATP adenosine triphosphate DI deionized EtOH ethanol GC gaschromatography MOPS 3-(N-morpholino)-propanesulfonic acid MTBE methyltert-butyl ether PDA photodiode array RRT relative retention time RTroom temperature THF tetrahydrofuran TMB 3,3′,5,5′-tetramethylbenzidine

EXAMPLES

The following examples illustrate the invention and are set forth to aidin the understanding of the invention, and are not intended and shouldnot be construed to limit in any way the invention set forth in theclaims which follow thereafter.

In the examples described below, unless otherwise indicated alltemperatures are set forth in degrees Celsius. Reagents were purchasedfrom commercial suppliers such as Sigma-Aldrich Chemical Company, EMD, JT Baker, or Pharco-Aaper, and were used without further purificationunless otherwise indicated. Tetrahydrofuran (THF), heptane and otherorganic solvents were purchased from commercial suppliers, such asSigma-Aldrich Chemical Company, ACROS, Alfa-Aesar, Lancaster, TCI, orMaybridge, and used as received.

One skilled in the art will recognize that, where not otherwisespecified, the reaction step(s) is performed under suitable conditions,according to known methods, to provide the desired product. One skilledin the art will also recognize that wherein a reaction step as disclosedherein may be carried out in a variety of solvents or solvent systems,said reaction step may also be carried out in a mixture of the suitablesolvents or solvent systems. One skilled in the art will recognize that,in the specification and claims as presented herein, wherein a reagentor reagent class/type (e.g. base, solvent, etc.) is recited in more thanone step of a process, the individual reagents are independentlyselected for each reaction step and may be the same of different fromeach other. For example wherein two steps of a process recite an organicor inorganic base as a reagent, the organic or inorganic base selectedfor the first step may be the same or different than the organic orinorganic base of the second step.

The reactions set forth below were done generally under a positivepressure of nitrogen (unless otherwise stated) in “ACS grade” solvents,and the reaction flasks were typically fitted with rubber septa for theintroduction of substrates and reagents via syringe or addition funnel.

Two reversed-phase high performance liquid chromatography (HPLC) systemswere used for in-process monitoring and analysis, using acetonitrile andwater/trifluoroacetic acid as mobile phases. One system employed anAgilent Zorbax Extend C18 column at 264 nm, while the other system(hereinafter, “TRK1PM1 HPLC”) included a Waters Xbridge Phenyl Column at268 nm. Unless otherwise specified, the former system was used. Thesilica for both systems was stirred in a flask with the compound, andthen filtered through a polypropylene cloth before being analyzed.

Amorphous Freebase Form of Compound of Formula (I):

About 1 gramof(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide is dissolved inminimum amount of water and cooled to a temperature of about

−26° Celsius followed by drying in the freeze dryer for 24 hours. About20 mg of the amorphous material obtained from the freeze dryer wasweighed in a vial, to which 5 volume aliquots of an appropriate solventsystem was added. The mixture was checked for dissolution and if nodissolution was apparent, the mixture was heated to about 40° Celsiusand checked again. This procedure was continued until dissolution wasobserved or until 100 volumes of solvent had been added. The XRPDpattern of the amorphous material obtained from the freeze dryingexperiment is shown in FIG. 7.

Amorphous hydrogen sulfate salt of compound of formula (I) was preparedas described in Example 14A in WO 2010/048314 (see Example 3). The XRPDpatterns of the two different lots of amorphous material prepared bythis method are show in FIG. 7.

Also provided herein is a process for the preparation of crystallineform (I-HS). In some embodiments, the process comprises the steps asshown in Scheme 1.

In some embodiments, provided herein is a process for the preparation ofcrystalline form (I-HS), comprising:

(a) adding concentrated sulfuric acid to a solution of(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidein EtOH to form the hydrogen sulfate salt of(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide;

(b) adding heptane to the solution in Step (a) to form a slurry;

(c) filtering the slurry to isolate(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehydrogen sulfate;

(d) mixing said(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehydrogen sulfate with a 5:95 w/w solution of water/2-butanone;

(e) heating the mixture from step (d) at about 65-70° C. with stirringuntil the weight percent of ethanol is about 0.5% to form a slurry ofthe crystalline form of(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehydrogen sulfate; and

(f) isolating the crystalline form of(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehydrogen sulfate by filtration.

In some embodiments, the above method further comprises: (b1) seedingthe solution from step (a) with(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-I-carboxamidehydrogen sulfate at room temperature and allowing the solution to stiruntil a slurry forms.

In some embodiments, provided herein is a process for the preparation ofcrystalline form (I-HS), comprising:

(a) reacting 5-chloro-3-nitropyrazolo[1,5-a]pyrimidine with(R)-2-(2,5-difluorophenyl)pyrrolidine (R)-2-hydroxysuccinate in thepresence of a base to form(R)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)-3-nitropyrazolo[1,5-a]pyrimidine;

(b) treating said(R)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)-3-nitropyrazolo[1,5-a]pyrimidinewith Zn and hydrochloric acid to form(R)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-amine;

(c) treating said(R)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-aminewith a base and phenyl chloroformate to form phenyl(R)-(5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)carbamate;

(d) reacting said phenyl(R)-(5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)carbamatewith (S)-pyrrolidin-3-ol to form(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide;

(e) adding sulfuric acid to said(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamideform(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehydrogen sulfate; and

(f) isolating the crystalline form of(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehydrogen sulfate.

In some embodiments of the above step (a), the base is an amine base,such as triethylamine.

In some embodiments of the above step (c), the base is an alkali metalbase, such as an alkali metal carbonate, such as potassium carbonate.

Preparation of 5-chloro-3-nitropyrazolo[1,5-a]pyrimidine StepA—Preparation of Sodium pyrazolo[1,5-a]pyrimidin-5-olate

A solution of 1H-pyrazol-5-amine and1,3-dimethylpyrimidine-2,4(1H,3H)-dione (1.05 equiv.) were charged to around bottom flask outfitted with a mechanical stirrer, a steam pot, areflux condenser, a J-Kem temperature probe and an N₂ adaptor forpositive N₂ pressure control. Under mechanical stirring the solids weresuspended with 4 vol. (4 mL/g) of absolute EtOH under a nitrogenatmosphere, then charged with 2.1 equivalents of NaOEt (21 wt % solutionin EtOH), and followed by line-rinse with 1 vol. (1 mL/g) of absoluteEtOH. The slurry was warmed to about 75° Celsius and stirred at gentlereflux until less than 1.5 area % of 1H-pyrazol-5-amine was observed byTRK1PM1 HPLC to follow the progression of the reaction using 20 μL ofslurry diluted in 4 mL deionized water and 5 μL injection at 220 nm.

After 1 additional hour, the mixture was charged with 2.5 vol. (2.5mL/g) of heptane and then refluxed at 70° Celsius for 1 hour. The slurrywas then cooled to room temperature overnight. The solid was collectedby filtration on a tabletop funnel and polypropylene filter cloth. Thereactor was rinsed and charged atop the filter cake with 4 vol. (4 mL/g)of heptane with the cake pulled and the solids being transferred totared drying trays and oven-dried at 45° Celsius under high vacuum untiltheir weight was constant. Pale yellow solid sodiumpyrazolo[1,5-a]-pyrimidin-5-olate was obtained in 93-96% yield(corrected) and larger than 99.5 area % observed by HPLC (1 mg/mLdilution in deionized water, TRK1PM1 at 220 nm).

Step B—Preparation of 3-nitropyrazolo[1,5-a]pyrimidin-5(4H)-one

A tared round bottom flask was charged with sodiumpyrazolo[1,5-a]pyrimidin-5-olate that was dissolved at 40-45° Celsius in3.0 vol. (3.0 mL/g) of deionized water, and then concentrated under highvacuum at 65° Celsius in a water-bath on a rotary evaporator until2.4×weight of starting material was observed (1.4 vol/1.4 mL/g deionizedwater content). Gas chromatography (GC) for residual EtOH (30 μL ofsolution dissolved in ˜1 mL MeOH) was performed showing less than 100ppm with traces of ethyl nitrate fumes being observed below upon lateraddition of HNO₃. In some cases, the original solution was charged withan additional 1.5 vol. (1.5 mL/g) of DI water, then concentrated underhigh vacuum at 65° Celsius in a water-bath on a rotary evaporator until2.4×weight of starting material was observed (1.4 vol/1.4 mL/g DI watercontent). Gas chromatograph for residual EtOH (30 μL of solutiondissolved in about 1 mL MeOH) was performed showing <<100 ppm ofresidual EtOH without observing any ethyl nitrate fumes below upon lateraddition of HNO₃.

A round bottom vessel outfitted with a mechanical stirrer, a steam pot,a reflux condenser, a J-Kem temperature probe and an N₂ adaptor forpositive N₂ pressure control was charged with 3 vol. (3 mL/g, 10 equiv)of >90 wt % HNO₃ and cooled to about 10° Celsius under a nitrogenatmosphere using external ice-water cooling bath under a nitrogenatmosphere. Using a pressure equalizing addition funnel, the HNO₃solution was charged with the 1.75-1.95 volumes of a deionized watersolution of sodium pyrazolo[1,5-a]pyrimidin-5-olate (1.16-1.4 mL DIwater/g of sodium pyrazolo[1,5-a]pyrimidin-5-olate) at a rate tomaintain 35-40° Celsius internal temperature under cooling. Twoazeotropes were observed without any ethyl nitrate fumes. The azeotropeflask, the transfer line (if applicable) and the addition funnel wererinsed with 2×0.1 vol. (2×0.1 mL/g) deionized water added to thereaction mixture. Once the addition was complete, the temperature wasgradually increased to about 45-50° Celsius for about 3 hours with HPLCshowing >99.5 area % conversion of sodiumpyrazolo[1,5-a]pyrimidin-5-olate to3-nitropyrazolo[1,5-a]pyrimidin-5(4H)-one.

Step C—Preparation of 5-chloro-3-nitropyrazolo[1,5-a]pyrimidine

3-nitropyrazolo[1,5-a]pyrimidin-5(4H)-one was charged to a round bottomflask outfitted with a mechanical stirrer, a heating mantle, a refluxcondenser, a J-Kem temperature probe and an N₂ adaptor for positive N₂pressure control. Under mechanical stirring the solids were suspendedwith 8 volumes (8 mL/g) of CH₃CN, and then charged with 2,6-lutitine(1.05 equiv) followed by warming the slurry to about 50° Celsius. Usinga pressure equalizing addition funnel, the mixture was dropwise chargedwith 0.33 equivalents of POCl₃. This charge yielded a thick, beigeslurry of a trimer that was homogenized while stirring until asemi-mobile mass was observed. An additional 1.67 equivalents of POCl₃was charged to the mixture while allowing the temperature to stabilize,followed by warming the reaction mixture to a gentle reflux (78°Celsius). Some puffing was observed upon warming the mixture that latersubsided as the thick slurry got thinner.

The reaction mixture was allowed to reflux until complete dissolution toa dark solution and until HPLC (20 μL diluted in 5 mL of CH₃CN, TRK1PM1HPLC, 5 μL injection, 268 nm) confirmed that no more trimer (RRT 0.92)was present with less than 0.5 area % of3-nitropyrazolo[1,5-a]pyrimidin-5(4H)-one (RRT 0.79) being observed bymanually removing any interfering and early eluting peaks related tolutidine from the area integration. On a 1.9 kg scale, 0 area % of thetrimer, 0.25 area % of 3-nitropyrazolo[1,5-a]pyrimidin-5(4H)-one, and99.5 area % of 5-chloro-3-nitropyrazolo[1,5-a]pyrimidine was observedafter 19 hours of gentle reflux using TRK1PM1 HPLC at 268 nm

Preparation of (R)-2-(2,5-difluorophenyl)pyrrolidine(R)-2-hydroxysuccinate Step A—Preparation of tert-butyl(4-(2,5-difluorophenyl)-4-oxobutyl)-carbamate

2-bromo-1,4-difluorobenzene (1.5 eq.) was dissolved in 4 volumes of THF(based on weight of tert-butyl 2-oxopyrrolidine-1-carboxylate) andcooled to about 5° Celsius. A solution of 2.0 M iPrMgCl in THF (1.4 eq.)was added over 2 hours to the mixture while maintaining a reactiontemperature below 250 Celsius. The solution was allowed to cool to about5° Celsius and stirred for 1 hour (GC analysis confirmed Grignardformation). A solution of tert-butyl 2-oxopyrrolidine-1-carboxylate (1.0eq.) in 1 volume of THF was added over about 30 min while maintaining areaction temperature below 25° Celsius. The reaction was stirred atabout 50 Celsius for 90 min (tert-butyl 2-oxopyrrolidine-1-carboxylatewas confirmed to be less than 0.5 area % by HPLC). The reaction wasquenched with 5 volumes of 2 M aqueous HCl while maintaining a reactiontemperature below 45° Celsius. The reaction was then transferred to aseparatory funnel adding 10 volumes of heptane and removing the aqueouslayer. The organic layer was washed with 4 volumes of saturated aqueousNaCl followed by addition of 2×1 volume of saturated aqueous NaCl. Theorganic layer was solvent-switched to heptane (<1% wt THF confirmed byGC) at a distillation temperature of 35-55° Celsius and distillationpressure of 100-200 mm Hg for 2×4 volumes of heptane being added with aminimum distillation volume of about 7 volumes. The mixture was thendiluted to 10 volumes with heptane while heating to about 55° Celsiusyielded a denser solid with the mixture being allowed to cool to roomtemperature overnight. The slurry was cooled to less than 5° Celsius andfiltered through polypropylene filter cloth. The wet cake was washedwith 2×2 volumes of heptane. The solids were dried under vacuum at 55°Celsius until the weight was constant, yielding tert-butyl(4-(2,5-difluorophenyl)-4-oxobutyl)-carbamate as a white solid at about75% to 85% theoretical yield.

Step B—Preparation of 5-(2,5-difluorophenyl)-3,4-dihydro-2H-pyrrole

tert-butyl (4-(2,5-difluorophenyl)-4-oxobutyl)-carbamate was dissolvedin 5 vol. of toluene with 2.2 eq. of 12M HCl being added observing amild exotherm and gas evolution. The reaction was heated to 65° Celsiusfor 12-24 hours and monitored by HPLC. Upon completion the reaction wascooled to less than 15° Celsius with an ice/water bath. The pH wasadjusted to about 14 with 3 equivalents of 2M aqueous NaOH (4.7 vol.).The reaction was stirred at room temperature for 1-2 hours. The mixturewas transferred to a separatory funnel with toluene. The aqueous layerwas removed and the organic layer was washed with 3 volumes of saturatedaqueous NaCl. The organic layer was concentrated to an oil andredissolved in 1.5 volumes of heptane. The resulting suspension wasfiltered through a GF/F filter paper and concentrated to a light yellowoil of 5-(2,5-difluorophenyl)-3,4-dihydro-2H-pyrrole with a 90% to 100%theoretical yield.

Step C—Preparation of (R)-2-(2,5-difluorophenyl)pyrrolidine

Chloro-1,5-cyclooctadiene iridium dimer (0.2 mol %) and(R)-2-(2-(diphenylphosphino)phenyl)-4-isopropyl-4,5-dihydrooxazole (0.4mol %) were suspended in 5 volumes of MTBE (based on5-(2,5-difluorophenyl)-3,4-dihydro-2H-pyrrole) at room temperature. Themixture was stirred for 1 hour and most of the solids dissolved with thesolution turning dark red. The catalyst formation was monitored using anHPLC/PDA detector. The reaction was cooled to less than 50 Celsius and5-(2,5-difluorophenyl)-3,4-dihydro-2H-pyrrole (1.0 eq.) was added usinga 0.5 volumes of MTBE rinse. Diphenylsilane (1.5 eq.) was added overabout 20 minutes while maintaining a reaction temperature below 10°Celsius. The reaction was stirred for 30 minutes below 10° Celsius andthen allowed to warm to room temperature. The reaction was stirredovernight at room temperature. The completion of the reaction wasconfirmed by HPLC and then cooled to less than 5° Celsius. The reactionwas quenched with 5 volumes of 2M aqueous HCl maintaining temperaturebelow 20° Celsius. After 10 minutes the ice/water bath was removed andthe reaction temperature was allowed to increase to room temperaturewhile stirring for 2 hours. The mixture was transferred to a separatoryfunnel with 3 volumes of MTBE. The aqueous layer was washed with 3.5volumes of MTBE followed by addition of 5 volumes of MTBE to the aqueouslayer while adjusting the pH to about 14 by adding 0.75 volumes ofaqueous 50% NaOH. The organic layer was washed with 5 volumes of aqueoussaturated NaCl, then concentrated to an oil, and diluted with 3 volumesof MTBE. The solution was filtered through a polypropylene filter clothand rinsed with 1 volume of MTBE. The filtrate was concentrated to anoil of (R)-2-(2,5-difluorophenyl)pyrrolidine with a 95% to 100%theoretical yield and with 75-85% ee.

Step D—Preparation of (R)-2-(2,5-difluorophenyl)pyrrolidine(R)-2-hydroxy-succinate

(R)-2-(2,5-difluorophenyl)pyrrolidine (1.0 eq.) was transferred to around bottom flask charged with 15 volumes (corrected for potency) ofEtOH (200 prf). D-malic acid (1.05 eq.) was added and the mixture washeated to 65° Celsius. The solids all dissolved at about 64° Celsius.The solution was allowed to cool to RT. At about 55° Celsius thesolution was seeded with (R)-2-(2,5-difluorophenyl)pyrrolidine(R)-2-hydroxy-succinate (about 50 mg, >97% ee) and stirred at roomtemperature overnight. The suspension was then filtered through apolypropylene filter cloth and washed with 2×1 volumes of EtOH (200prf). The solids were dried under vacuum at 55° Celsius, yielding(R)-2-(2,5-difluorophenyl)pyrrolidine (R)-2-hydroxy-succinate with a 75%to 90% theoretical yield and with >96% ee.

Referring to Scheme 1, suitable bases include tertiary amine bases, suchas triethylamine, and K₂CO₃. Suitable solvents include ethanol, heptaneand tetrahydrofuran (THF). The reaction is conveniently performed attemperatures between 5° Celsius and 50° Celsius. The reaction progresswas generally monitored by HPLC TRK1PM1.

Compounds II (5-chloro-3-nitropyrazolo[1,5-a]pyrimidine) and III((R)-2-(2,5-difluorophenyl)pyrrolidine (R)-2-hydroxysuccinate, 1.05 eq.)were charged to a round bottom flask outfitted with a mechanicalstirrer, a J-Kem temperature probe and an N₂ adaptor for positive N₂pressure control. A solution of 4:1 EtOH:THF (10 mL/g of compound II)was added and followed by addition of triethylamine (NEt₃, 3.50 eq.) viaaddition funnel with the temperature reaching about 40° Celsius duringaddition. Once the addition was complete, the reaction mixture washeated to 50° Celsius and stirred for 0.5-3 hours to yield compound IV.

To a round bottom flask equipped with a mechanical stirrer, a J-Kemtemperature probe, and an N₂ inlet compound IV was added and followed byaddition of tetrahydrofuran (10 mL/g of compound IV). The solution wascooled to less than 5° Celsius in an ice bath, and Zn (9-10 eq.) wasadded. 6M HCl (9-10 eq.) was then added dropwise at such a rate to keepthe temperature below 30° Celsius (for 1 kg scale the addition tookabout 1.5 hours). Once the exotherm subsided, the reaction was allowedto warm to room temperature and was stirred for 30-60 min until compoundIV was not detected by HPLC. At this time, a solution of potassiumcarbonate (K₂CO₃, 2.0 eq.) in water (5 mL/g of compound IV) was addedall at once and followed by rapid dropwise addition of phenylchloroformate (PhOCOCl, 1.2 eq.). Gas evolution (CO₂) was observedduring both of the above additions, and the temperature increased toabout 30° Celsius after adding phenyl chloroformate. The carbamateformation was stirred at room temperature for 30-90 min. HPLC analysisimmediately followed to run to ensure less than 1 area % for the aminebeing present and high yield of compound VI in the solution.

To the above solution amine VII ((S)-pyrrolidin-3-ol, 1.1 eq. based ontheoretical yield for compound VI) and EtOH (10 mL/g of compound VI) wasadded. Compound VII was added before or at the same time as EtOH toavoid ethyl carbamate impurities from forming. The above EtOH solutionwas concentrated to a minimum volume (4-5 mL/g) using the batchconcentrator under reduced pressure (THF levels should be <5% by GC),and EtOH (10 mL/g of compound VI) was back-added to give a total of 10mL/g. The reaction was then heated at 50° Celsius for 9-19 hours oruntil HPLC shows that compound VI is less than 0.5 area %. The reactionwas then cooled to room temperature, and sulfuric acid (H₂SO₄, 1.0 eq.to compound VI) was added via addition funnel to yield compound I-HSwith the temperature usually exotherming at about 30° Celsius.

Example 1 Preparation of Crystalline Form (I-HS) (Method 1)

(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide(0.500 g, 1.17 mmol) was dissolved in EtOH (2.5 mL) and cooled to about5° Celsius. Concentrated sulfuric acid (0.0636 mL, 1.17 mmol) was addedto the cooled solution and stirred for about 10 min, while warming toroom temperature. Methyl tert-butyl ether (MTBE) (2 mL) was slowly addedto the mixture, resulting in the product gumming out. EtOH (2.5 mL) wasthen added to the mixture and heated to about reflux until all solidswere dissolved. Upon cooling to room temperature and stirring for about1 hour, some solids formed. After cooling to about 5° Celsius, thesolids were filtered and washed with MTBE. After filtration and dryingat air for about 15 minutes,(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehydrogen sulfate was isolated as a solid.

Example 2 Preparation of Crystalline Form (I-HS) (Method 2)

Concentrated sulfuric acid (392 mL) was added to a solution of 3031 g of(S)—N-(5-((R)-2-(2, 5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide in 18322 mL EtOHto form the hydrogen sulfate salt. The solution was seeded with 2 g of(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehydrogen sulfate and the solution was stirred at room temperature for atleast 2 hours to form a slurry of the hydrogen sulfate salt. Heptane(20888 g) was added and the slurry was stirred at room temperature forat least 60 min. The slurry was filtered and the filter cake was washedwith 1:1 heptane/EtOH. The solids were then dried under vacuum atambient temperature (oven temperature set at 15° Celsius).

The dried hydrogen sulfate salt (6389 g from 4 combined lots) was addedto a 5:95 w/w solution of water/2-butanone (total weight 41652 g). Themixture was heated at about 68° Celsius with stirring until the weightpercent of ethanol was about 0.5%, during which time a slurry formed.The slurry was filtered, and the filter cake was washed with a 5:95 w/wsolution of water/2-butanone. The solids were then dried under vacuum atambient temperature (oven temperature set at 15° Celsius) to provide thecrystalline form of(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehydrogen sulfate.

Example 3 Preparation of Amorphous Form AM(HS)

To a solution of(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide(9.40 g, 21.94 mmol) in MeOH (220 mL) was slowly added sulfuric acid(0.1 M in MeOH, 219.4 mL, 21.94 mmol) at ambient temperature under rapidstirring. After 30 minutes, the reaction was first concentrated byrotary evaporator to near dryness, then on high vacuum for 48 h toprovide amorphous form of(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidesulfate (11.37 g, 21.59 mmol, 98.43% yield). LCMS (apci m/z 429.1, M+H).

Example 4 Preparation of Crystalline HCl Salt of Formula (I)

A mixture of(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide(0.554 g, 1.29 mmol) in EtOH (6 mL, 200 proof) and MTBE (10 mL) washeated to 50° C. while stirring to obtain a solution, followed byaddition of hydrogen chloride (conc.) (0.108 mL, 1.29 mmol) in oneportion. The reaction mixture was then allowed to cool to ambienttemperature first, then cooled to about 5° C. in an ice-water bath withstirring to induce crystallization. The suspension was stirred for 4 hin the ice-water bath before it was vacuum-filtered, with the filtercake rinsed with MTBE and dried under vacuum at 55° C. to constantweight, yielding crystalline(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehydrochloride (0.534 g, 89% yield). LCMS (apci m/z 429.2, M+H).

Preparation of Crystalline HBr Salt of Formula (I)

A mixture of(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide(0.505 g, 1.18 mmol) in EtOH (6 mL, 200 proof) and MTBE (10 mL) washeated to 50° C. while stirring to obtain a solution, followed byaddition of hydrogen bromide (33% aq.) (0.213 mL, 1.18 mmol) in oneportion. The reaction mixture was heated to reflux to obtain a mostlyclear solution with small amount of oily residue on glass wall ofreaction vessel. Upon cooled to ambient temperature, precipitationappeared and the oily residue solidified. The mixture was heated to 50°C. again, then allowed to cool to room temperature and stirred forovernight. The suspension was vacuum-filtered, with the filter cakerinsed with MTBE and dried under vacuum at 55° C. to constant weight,yielding crystalline(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-I-carboxamidehydrobromide (0.51 g, 85% yield). LCMS (apci m/z 429.3, M+H).

Preparation of Crystalline Mesylate Salt of Formula (I)

A mixture of(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide(0.532 g, 1.24 mmol) in EtOH (2.7 mL, 200 proof) and MTBE (5.3 mL) washeated to 50° C. while stirring to obtain a solution, followed byaddition of methanesulfonic acid (0.076 mL, 1.24 mmol) in one portion.The reaction mixture was heated to reflux to obtain a mostly clearsolution with small amount of particulates. Upon cooled to ambienttemperature, precipitation appeared along with some oily residue.Additional EtOH (0.5 mL, 200-proof) and methanesulfonic acid (0.010 mL)were added to obtain a solution. The reaction mixture was heated to 50°C. again, then allowed to cool to room temperature and stirred for 1 h.The suspension was vacuum-filtered, with the filter cake rinsed withMTBE and dried under vacuum at 55° C. to constant weight, yieldingcrystalline(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidemethanesulfonate (0.51 g, 78% yield). LCMS (apci m/z 429.4, M+H).

Preparation of Crystalline Camsylate Salt of Formula (I)

A mixture of(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide(0.500 g, 1.17 mmol) and S-(+)-camphorsulfonic acid (0.271 g, 1.17 mmol)in EtOH (3 mL, 200 proof) and MTBE (5 mL) was heated to reflux whilestirring to obtain a solution. Upon cooled to ambient temperature,precipitation appeared. The suspension was stirred at room temperaturefor overnight, then vacuum-filtered, with the filter cake rinsed withMTBE and dried under vacuum at 55° C. to constant weight, yieldingcrystalline(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide((1 S,4R)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptan-1-yl)methanesulfonate.

Example 5 Infantile Fibrosarcoma with NTRK3-ETV6 Fusion SuccessfullyTreated with a Liquid Formulation of(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamideMaterials and Methods

A multicenter pediatric phase 1 dose-escalation study in patients withadvanced solid or primary CNS tumors was initiated in December 2015(ClinicalTrials.gov Identifier: NCT02637687) to evaluate the safety andtolerability of Compound I-HS (i.e., the hydrogen sulfate salt of(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide).Eligibility criteria included age 1-21 years regardless of the presenceof a known TRK alteration, as well as those patients aged 1 month of ageor greater with a known NTRK fusion and a diagnosis of infantilefibrosarcoma or congential mesoblastic nephroma. An oral liquidformulation of Compound I-HS was developed for patients unable toswallow capsules. SIMCYP® Pediatric Simulation modeling (CERTARA,Princeton, N.J.) was utilized to establish a pharmacokinetic approachfor dosing that takes into account patient age, ontogeny of theclearance pathways that eliminate Compound I-HS, and body surface area(BSA). The pediatric dose selected for the initial cohort was predictedto equal the exposure achieved in adult patients taking a dose of 100 mgBID, the recommended Phase 2 adult dose. Cycles are measured in 28-dayincrements with continuous dosing. Response assessments by appropriateimaging modalities are scheduled every eight weeks. Patients continue ontherapy until evidence of disease progression or intolerable toxicity.

A kit was provided that included a sealed graduated amber bottlecontaining 7.6 g of Compound I-HS; a sealed bottle containing 51 gCAVASOL® W7 HP Pharma; a sealed bottle containing 500 g trisodiumcitrate dihydrate; a sealed bottle containing 100 mL sterile water; asealed pint (˜473 mL) bottle of ORA-Sweet® SF; a funnel; a 28-mmpress-in bottle adaptor; a box containing 56 units of 1-mL single usedosing syringes; a box containing 56 units of 5-mL single use dosingsyringes; a drug product label indicating the concentration of CompoundI-HS (20 mg/mL); and compounding instructions.

A liquid solution was prepared as shown in FIG. 9. First, the seal (cap)was removed from the bottle containing CAVASOL® W7 HP Pharma. Next,using the funnel, the contents of the 100 mL bottle of sterile water towere added to the bottle containing CAVASOL® W7 HP Pharma. The bottlewith its cap was then closed and the bottle containing CAVASOL® W7 HPPharma and sterile water was shaken until all of the CAVASOL® W7 HP wasdissolved. Ten minutes was allowed to pass for full dissolution of theCAVASOL® W7 HP Pharma. The bottom and sides of the bottle were inspectedto make sure all CAVASOL® W7 HP Pharma dissolved and was not clumped onthe bottom or clinging to the sides. Next, the bottle was allowed tostand without agitation for approximately five minutes to allow thebubbles created from dissolved CAVASOL® W7 HP Pharma to dissipate. Theseal (cap) from the graduated bottle containing Compound I-HS was thenremoved. Using the same funnel from earlier, the CAVASOL® W7 HP Pharmasolution was added to the graduated bottle containing Compound I-HS. Thebottle was capped and shaken by hand until dissolved. Bubbles wereallowed to come to surface and a clear red solution resulted. Using thesame funnel from earlier, q.s. to 300 mL with the supplied ORA-Sweet®SF. The graduated bottle was capped and gently inverted 10 times to mixthe ORA-Sweet® SF with the Compound I-HS/CAVASOL® W7 HP solution whilebeing careful not to introduce too many bubbles into the formulation.Next, 3.5 g trisodium citrate dihydrate from the provided container ofTrisodium Citrate Dihydrate was weighed and added, using the secondfunnel in the kit, to the liquid formulation and, subsequently, thebottle was capped and the bottle was inverted ten times. The bubbleswere allowed to rise to the top and the contents of the bottle wereinspected to make sure all of the trisodium citrate dihydrate was fullydissolved; if it was not, the bottle was inverted an additional 10times. Subsequently, the cap on the graduated bottle was removed and theprovided 28-mm press-in bottle adaptor (syringe adaptor) was inserted inthe bottle. The bottle was then closed by securely placing the cap onthe bottle. The liquid formulation was then administered the desiredamount of Compound I-HS using a 1 mL or 5 mL syringe, depending onpatient dosing regimen.

Results

An otherwise healthy female was born with a large, vascular, right-sidedneck mass extending to the face that was initially diagnosed and treatedas a Rapidly Involuting Congenital Hemangioma. At 6 months of age, themass grew rapidly and surgical excision/debulking revealed the diagnosisof IFS confirmed by an ETV6 translocation by fluorescent in situhybridization (FISH). Within the first 7 days post-operatively, thetumor rapidly progressed, encroaching the oral cavity. Chemotherapy withvincristine, actinomycin-D and cyclophosphamide was initiated but thepatient experienced disease progression during cycle 1. A newchemotherapy regimen comprised of ifosfamide and doxorubicin (ID) wasstarted concurrently with debulking surgery and a tracheostomy wasplaced-for oropharyngeal obstruction. Two additional courses of ID andfour courses of ifosfamide and etoposide had minimal impact on thetumor. The tumor progressed to involve the base of skull, mastoids andcervical vasculature. Gross surgical resection was performed in October2015 by a team of multidisciplinary surgeons but clear surgical marginscould not be achieved.

Five weeks following surgical resection, an MR of the brain and neckshowed a 20 mm×19 mm×18 mm hyperenhancing mass involving the skull baseof the middle cranial fossa, just anterior and inferior to the inner earstructures (see FIG. 10A and FIG. 10B). Further chemotherapy wasdetermined to be futile due to lack of response to all standardregimens. Repeat surgical resection was deemed not possible. Therapeuticradiotherapy was possible, but based on the age of the patient andlocation of the disease, it was expected to produce devastatinglong-term sequalae.

In December 2015, at the age of 16 months, the patient enrolled on thePhase 1 pediatric study of the oral, selective TRK inhibitor CompoundI-HS. The parents noted improved engagement and playfulness throughoutcycle 1. At the end of cycle 1 (day 28), an MR of the brain and neckshowed a significant interval reduction in the size and enhancement ofthe mass by more than 90% from baseline (see FIG. 10C and FIG. 10D).Repeat scans at the end of Cycle 2 confirmed the size reduction andshowed continued decrease in enhancement, confirming partial response(see FIG. 10E and FIG. 10F). During the first two cycles, the patientexperienced fever and PCR-confirmed influenza A (considered not related)but no adverse events related to Compound I-HS.

Example 6 A Liquid Formulations of(S)—N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide

A liquid formulation of(S)—N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidewas prepared with the components listed in Table 16.

TABLE 16 A liquid formulations of(S)-N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide. TotalTheoretical Formulation Quantity % Weight Weight in grams RequiredAmount per Material Name (a) (b) (a × b)/100 bottle⁽¹⁾ Compound I-HS API2.05% 171,648 3,518.8 grams⁽¹⁾⁽²⁾ 1.47 g Purified Water, USP 33.55%57,587.9 grams 24.01 g  KLEPTOSE ® HPB 14.55% 24,974.8 grams 10.48 g Parenteral Grade EP, USP ORA-SWEET ® 48.51% 83,266.4 grams 34.93 g Sodium Citrate, Dihydrate, 0.94% 1,613.5 grams 0.68 g Granular, USP(Spectrum) (1,694.2 grams)⁽³⁾ 231a12 Natural Masking 0.10% 171.6 grams0.07 g Type Flavor (Abelei) 231a39 Natural Bitterness 0.20% 343.3 grams0.14 g Masking Type Flavor (Abelei) Bitterness Masking Flavor, 0.05%85.8 grams 0.04 g Nat (FONA - Liquid) FONATECH ® Taste 0.05% 85.8 grams0.04 g Modifier Flavor, Nat ⁽¹⁾Includes an API correction factor of0.8137. Calculation: Free base molecular weight/salt formula weight =428.44/526.51. Density of the liquid formulation is 1.2 mg/mL. ⁽²⁾Labelclaim = 3,518.8 grams Salt Form API x 0.8137/171,648 grams totalformulation * 1.2 g/mL density * 1,000 mg/g. ⁽³⁾Includes an additional5% of the total amount of Sodium Citrate added to the formulation for pHadjustment, as needed.

REFERENCES

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What is claimed:
 1. A method of treating a pediatric cancer in a subjectin need thereof, wherein the cancer is selected from the groupcomprising congenital mesoblastic nephroma, Ph-like acute lymphoblasticleukemia, infantile fibrosarcoma, pediatric high-grade glioma (HGG),diffuse intrinsic pontine gliomas (DIPGs), pediatric papillary thyroidcarcinoma, soft tissue sarcoma, and spindle cell sarcoma, the methodcomprising administering to the subject a therapeutically effectiveamount of(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehaving the formula (I):

a pharmaceutically acceptable salt thereof, or combinations thereof. 2.The method of claim 1, wherein the subject is an infant.
 3. The methodof claim 1, wherein the cancer is mediated by TrkA, TrkB, TrkC, orcombinations thereof.
 4. The method of claim 1, wherein the pediatriccancer is infantile fibrosarcoma.
 5. The method of claim 4, whereinsurgical resection has failed to inhibit progression of the fibrosarcomain the subject.
 6. The method of claim 1, wherein chemotherapypreviously failed to inhibit tumor progression in the subject.
 7. Themethod of claim 1, further comprising performing one or more ofmorphological diagnosis and molecular testing prior to administering thecompound of formula (I), a pharmaceutically acceptable salt thereof, ora combination thereof.
 8. The method of claim 1, wherein the compound offormula (I) is a hydrogen sulfate salt.
 9. The method of claim 1,wherein the compound of formula (I) is provided as a crystalline form.10. The method of claim 1, wherein the compound of formula (I), apharmaceutically acceptable salt thereof, or a combination thereof, isprovided as a liquid formulation.
 11. The method of claim 10, whereinthe liquid formulation further comprises: a solubilizing agent, whereinthe solubilizing agent comprises a 3-cyclodextrin; and a base; wherein:the formulation has a pH of about 2.5 to about 5.5; and the compound offormula (I) has a concentration of about 15 mg/mL to about 35 mg/mL inthe liquid formulation.
 12. The method of claim 11, wherein theformulation has a pH of about 3 to about
 4. 13. The method of claim 11,wherein the β-cyclodextrin is hydroxypropyl-β-cyclodextrin.
 14. Themethod of claim 11, wherein the base is a citrate.
 15. The method ofclaim 14, wherein the base comprises sodium citrate dihydrate.
 16. Themethod of claim 10, wherein the liquid formulation further comprises: asolubilizing agent, wherein the solubilizing agent comprises aβ-cyclodextrin; a base; a sweetener; a bitterness masking agent; and aflavoring agent, wherein: the formulation has a pH of about 2.5 to about5.5; and the compound of formula (I) has a concentration of about 15mg/mL to about 35 mg/mL in the liquid formulation.
 17. The method ofclaim 10, wherein the liquid formulation is prepared from a crystallineform of the compound of formula (I) having the formula (I-HS):


18. The method of claim 17, wherein the crystalline form ischaracterized by having XRPD diffraction peaks (2θ degrees) at 18.4±0.2,20.7±0.2, 23.1±0.2, and 24.0±0.2.
 19. The method of claim 17, whereinthe crystalline form is characterized by having XRPD diffraction peaks(2θ degrees) at 10.7±0.2, 18.4±0.2, 20.7±0.2, 23.1±0.2, and 24.0±0.2.20. The method of claim 17, wherein the crystalline form ischaracterized by having XRPD diffraction peaks (2θ degrees) at 10.7±0.2,18.4±0.2, 19.2±0.2, 20.2±0.2, 20.7±0.2, 21.5±0.2, 23.1±0.2, and24.0±0.2.
 21. The method of claim 17, wherein the crystalline form ischaracterized by having XRPD diffraction peaks (2θ degrees) at 10.7±0.2,15.3±0.2, 16.5±0.2, 18.4±0.2, 19.2±0.2, 19.9±0.2, 20.2±0.2, 20.7±0.2,21.5±0.2, 22.1±0.2, 23.1±0.2, 24.0±0.2, 24.4±0.2, 25.6±0.2, 26.5±0.2,27.6±0.2, 28.2±0.2, 28.7±0.2, 30.8±0.2, and 38.5±0.2.
 22. The method ofclaim 1, wherein the compound of formula (I), a pharmaceuticallyacceptable salt thereof, or a combination thereof, is administered in28-day cycles.
 23. The method of claim 1, wherein the compound isadministered in a dosage calculated to be the equal to the exposure ofan adult taking the compound of formula (I), a pharmaceuticallyacceptable salt thereof, or a combination thereof, at a dose of 100 mgtwice a day.
 24. The method of claim 1, wherein the pediatric cancer isassociated with or having a dysregulation of a NTRK gene, a Trk protein,or expression or activity, or level of the same.
 25. The method of claim24, wherein the dysregulation of a NTRK gene, a Trk protein, orexpression or level of the same is a chromosome translation that resultsin the translation of a Trk fusion protein.
 26. The method of claim 25,wherein the Trk fusion protein is selected from the group consisting ofETV6-NTRK3, RET/NTRK1, TPM3-NTRK1, BTBD1-NTRK3, VCL-NTRK2, AGBL4-NTRK2,LMNA-NTRK1, TFG-NTRK1, QKI-NTRK2, NACC2-NTRK2, TPR-NTRK 1,RABGAP1L-NTRK1, MPRIP-NTRK1, SQSTM1-NTRK1, EML4-NTRK3, and AFAP1-NTRK2.27. The method of claim 26, wherein the Trk fusion protein isETV6-NTRK3.
 28. The method of claim 24, wherein the dyregulation of aNTRK gene, a Trk protein, or expression or activity of the same is oneor more point mutations in the gene.
 29. The method of claim 28, whereinthe one or more point mutations in the gene is one or more of (i) a TrkApoint mutation selected from the group consisting of C6773T, C7232T,C7301T, R33W, A336E, A337T, R324Q, R324W, V420M, R444Q, R444W, G517R,G517V, K538A, V573M, F589L, G595R, G667C, F598L, R649W, R649L, R682S,V683G, R702C, Q627X, Q597X, and Q633X; (ii) a TrkB point mutationselected from the group consisting of A13T, E142K, R136H, V619M, F633L,G639R, G709C, G709A, and G709S; and/or (iii) a TrkC point mutationselected from the group consisting of V603M, F617L, G623R, G696C, G696A,and G696S.